Cysteine-Rich Domains of Muc3 Intestinal Mucin Promote Cell Migration, Inhibit Apoptosis, and Accelerate Wound Healing

GASTROENTEROLOGY 2006;131:1501–1517

Cysteine-Rich Domains of Muc3 Intestinal Mucin Promote Cell Migration, Inhibit Apoptosis, and Accelerate Wound Healing SAMUEL B. HO,*,‡ LEAH A. DVORAK,* RACHEL E. MOOR,* AMANDA C. JACOBSON,* MARK R. FREY,§ JULISSA CORREDOR,§ D. BRENT POLK,§ and LAURIE L. SHEKELS*

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 ␣ 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.

M

ucins are a family of large secreted and membranebound glycoproteins expressed by most epithelial tissues, where they are directed to the cell surface and are thought to play a protective role.1,2 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. Membranebound 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.2 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.3,4 Posttranslational processing of membranebound 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.5,6 The mouse Muc3 mucin contains a cysteine-rich extracellular region with 2 EGF-like domains,7 and is similar in sequence and chromosomal localization to the human MUC3A, MUC3B, and MUC17 genes.3,4,8 Other membrane-bound mucins that contain EGF-like domains include human MUC49; the rodent homologue of MUC4, termed sialomucin complex, or ascites sialoglycoprotein ASGP1,2,10 MUC12,11 and MUC13.12 The MUC1 membranebound mucin is ubiquitous in all epithelial tissues but lacks cysteine-rich extracellular regions.2 The function of membranebound 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 Abbreviations used in this paper: BSA, bovine serum albumin; DSS, dextran sodium sulfate; EGF, epidermal growth factor; FCS, fetal calf serum; GST, glutathione S-transferase; MUC, mucin; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; TFF, Trefoil Factor Family; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate biotin nick-end labeling. © 2006 by the AGA Institute 0016-5085/06/$32.00 doi:10.1053/j.gastro.2006.09.006

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*Department of Medicine, Veterans Affairs Medical Center and University of Minnesota, Minneapolis, Minnesota; ‡Department of Medicine, Veterans Affairs San Diego Healthcare System and University of California, San Diego, California; and the §Department of Pediatrics and Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee

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survey as a possible site for a genetic susceptibility to inflammatory bowel disease.13,14 Kyo et al15 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.15 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,16 and mouse Muc3 and human MUC3A promoter activity can be regulated by cytokines and growth factors.17,18 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.19 We hypothesized that the cysteinerich 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.20 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.

Materials and Methods GST-Fusion Proteins 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.7 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-␤-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.

Cell Culture 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

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Type Culture Collection (Manassas, VA). A431 cells express high levels of EGF (ErbB1) receptor and migrate in response to EGF.21,22 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.23,24 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.3 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 ␮g 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 ␮g streptomycin/mL, as described previously.25,26 YAMC cells lack Muc3 mRNA as measured by reverse-transcription PCR (data not shown).

Cell Migration Assays 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.27 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 ␮mol/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.26 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

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A

MUC3 MUCIN CYSTEINE–RICH PROTEINS

. mouse muc3 EGF1,2 C-x10-C-x-C-x8-C-x8-C-x10-C-x-C-x8-C-x120-C-x4-C-x21-C-x22-C-x3-C-x9-C-x4-C-x8-C-x-C-x12C-X4

D

flag tag

EGF1

Linker region

EGF2

1503

transcytoplasmic membrane

human MUC3A EGF1,2 C-x10-C-x-C-x8-C-x5-C-x10-C-x-C-x8-C-x114-C-x6-C-x21-C-x22-C-x3-C-x9-C-x4-C-x8-C-x-C-x12C-x7 human MUC17 EGF1,2 C-x4-C-x6-C-x10-C-x-C-x8-C-x120-C-x4-C-x21-C-x21-C-x3-C-x9-C-x4-C-x8-C-x-C-x12-C EGF C-x7-C-x5-C-x10-C-x-C-x8—C trefoil C-X9-C-X9-C-X4—C-C-X10-C-X8-C

B

E

EGF1 domain: Human MUC3A Mouse Muc3

CDNGGTWEQGQCACLPGFSGDRCQL---QTRCQNGGQWDGLKCQCPSTFYGSSC CMNGGFWTGDKCICPNGFGGDRCENIVNVVNCENGGTWDGLKCQCTSLFYGPRCN

m3EGF1,2 m3EGF2

m3EGF1

GST

75 kDa

Linkage domain: Human MUC3A EFAVEQVDLDVVETEVGMEVSV---DQQFSPDLNDNTSQAYRDFNKTFWNQMQK IFADMQGFTFKGVEILSLRNGSIVVDYLVLLEMPFSPQLESEYEQVKTTLKEGL QNAS----QDVNS

50 kDa

Mouse Muc3 EELVESVEIEPTVA-ASVGVSVTVTSQEYSEKLQDRKSEEFSNFNKTFTKQMAL GVIIKNLSKGSIVVDYDVILKAKYTPGFENTLDTVVKNLETKI IYAGIPEYE— KNATEVQVQDVNNN

EGF2 domain: human MUC3A

35 kDa

CQDSQTLCFKPDSIKVNNNSKTELTPAAICRRAAPTGYEEFYFPLVEATRLRCVTKCTSGVDNAIDCHQGQCVLETSGPTCRCYSTDTHWFSGPRCEVAVHWR C--SALLCFNSTATKVQNSATVSVNPEETCKKEAGEDFAKFVTLGQKGDKWFCITPCSAGYSTSKNCSYGKCQLQRSGPQCLCLITDTHWYSGENCDWGIQKS

30 kDa 25 kDa

C

GST

EGF1

Linker

EGF2

m3EGF1,2 637

913

m3EGF1 637

690

m3EGF2 GST tag

810

913

Figure 1.

Structure of membrane-bound mucins and constructs. (A) Spacing of cysteines in the cysteine-rich region of mouse Muc3 and human MUC3A and MUC17. Cysteine spacing of EGF and trefoil motifs shown for comparison. Note the highly conserved cysteine arrangement in the EGF-like domains of mouse Muc3 and human MUC3A. The first and second EGF-like domains of Muc3 have 8 and 10 cysteines, respectively. The last 6 cysteines in each EGF-like domain are found in a spatial arrangement similar to EGF with the second EGF-like domain showing less conservation of the spacing. No other significant sequence similarity is found between the Muc3 EGF-like domains and EGF. (B) Amino acid sequence of the EGF1 domain, the glycosylated linkage domain, and the EGF2 domain of mouse Muc3 and human MUC3A. Human and mouse Muc3 share 60% and 44% overall sequence similarity between their first and second EGF-like domains, respectively. Comparison of the cysteine spacing of mouse Muc3 and human MUC17 shows less similarity, although the overall amino acid sequence similarity of mouse Muc3 and human MUC17 is comparable with the similarity with human MUC3A (52% and 64% sequence similarity in the first and second EGF-like domains, respectively, not shown). (C) Diagram of recombinant mouse GST-Muc3 fusion proteins expressed and purified from E coli. Numbers correspond to the amino acid numbering in the original Muc3 cDNA sequence described previously.7 (D) The 936-bp human MUC3A EGF1,2 construct encodes the 2 human MUC3A EGF-like domains, the MUC3A transmembrane region, and 20 amino acids of the MUC3A cytoplasmic domain. (E) Coomassie-stained SDS polyacrylamide gel electrophoresis of purified GST-labeled proteins. Lanes: m3EGF1,2, m3EGF1, m3EGF2, and GST.

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.

RNA Interference and Transfection MUC3A expression was knocked down using a small interfering RNA (siRNA) technique.28 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)

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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.

Cell Proliferation Assays

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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.25 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.29

Preparation of Cellular Lysates and Membranes 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 ␤-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 ␮L of RIPA lysis buffer and sheared using a 28-gauge needle. Reagents were purchased from Sigma.

Immunoprecipitation and Immunoblotting 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 ␮L/300 ␮L 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,

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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).

Thiol Quantification in Recombinant Peptides Determination of free cysteines in recombinant mucin proteins was performed using a method modified from Singh et al,30 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).

Stable Transfection of Human MUC3A Cysteine-Rich Domain Construct 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 ␮g/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 containing 800 ␮g/mL G418. Expression of the human MUC3A EGF1,2 construct was determined by Western analysis with rabbit anti-flag antibody (Sigma).

Apoptosis Assays Apoptosis was induced by adding 100 ng/mL of tumor necrosis factor (TNF)-␣ (Sigma) to subconfluent cultures of LoVo cells in 35-mm sterile Petri dishes in Dulbecco’s modified Eagle medium with 10% serum for 48 hours. Apoptosis also was induced by incubating cells with interferon-␥ 100 ng/mL for 24 hours, followed by removal of the interferon and the addition of anti-Fas antibody at 500 ng/mL for 48 hours (R&D Systems, Minneapolis, MN).31 The storage shelf-life of the interferon-␥ and anti-Fas antibody determined by the manufacturer was 3 months, and we observed a consistent decrease in apoptosis induced over time within this time period, which is reflected in differences in the maximal amount of apoptosis induced in each experiment. To account for these differences positive and negative controls were used in each experiment, consisting of cells with and without anti-Fas, and cells with and without recombinant mucin proteins or purified human recombinant EGF. Cells were pretreated with or without recombinant GSTfusion proteins 1 hour before addition of anti-Fas. For inhibition experiments, cells were pretreated with the EGF receptor inhibitor, tyrphostin (AG1478), when indicated, for 1 hour in fresh media followed by treatment with recombinant GSTfusion proteins 1 hour before the addition of anti-Fas. Cells were fixed in 4% paraformaldehyde in PBS pH 7.4 for 5 minutes, then washed twice in PBS. The cells were stained with the nuclear dye, Hoechst 33258 (Polysciences Inc., Warrington, PA), at a concentration of 5 ␮g/mL in PBS for 30 minutes, rinsed, cover-slipped with Slowfade Antifade (Molecular Probes), and then immediately imaged using an ultraviolet microscope. Apoptotic nuclei were identified by morphology.32 The total number of normal and apoptotic nuclei were counted in three 40⫻ lens fields per dish (representing ⬎200 nuclei per dish). Three or more dishes were used for each experimental condition. For consistent comparisons between trials, the data are represented as the percentage change in apoptosis from baseline (baseline is equal to the basal level of apoptosis that occurred in each assay and ranged from 9% and 3.9% between assays). Apoptosis was measured in histologic specimens using a terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) assay (TACS.XL In Situ Apoptosis Detection Kit; R&D Systems), according to the manufacturer’s directions. Positive controls consisted of sections of a human colorectal adenoma and negative controls consisted of incubating representative sections without the terminal deoxynucleotidyl transferase enzyme in the labeling reaction mixture, which resulted in negative staining. The number of apoptotic nuclei was counted in each low-power (10⫻ objective) field of distal colon specimens and the total number of apoptotic cells/distal colon was calculated for each specimen.

Experimental Colitis Models All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Minneapolis Veterans Affairs Medical Center.

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Acetic acid colitis. Female CD-1 mice (20 –30 g; Harlan Sprague Dawley, Indianapolis, IN) were fasted overnight and anesthetized with 3% isoflurane by inhalation. The rectum then was lavaged with .2 mL normal saline. Colitis was induced by intrarectal administration of .1 mL of 5% acetic acid. The solutions were administered through a trocar needle approximately 3 cm proximal to the anus. Mice subsequently were treated 12 and 24 hours later by intrarectal administration of .1 mL recombinant peptide in PBS or with .1 mL of control peptide in the same buffer at a similar concentration using isofluorane anesthesia. Then all mice were harvested at 30 hours after induction of colitis (6 hours after last treatment enema), and the distal colons were removed and examined for gross ulceration and microscopic examination. This model has been described previously.33,34 Dextran sodium sulfate colitis. Acute colitis was induced in female CD-1 mice (20 –30 g) by administration of 5% dextran sodium sulfate (molecular weight, 40,000 –50,000; USB, Cleveland, OH) in drinking water, as previously described.35–37 After 7 days the dextran sodium sulfate (DSS) was removed from the drinking water. Mice were treated 24 and 48 hours after removal of DSS by intrarectal administration of .1 mL recombinant peptide in PBS or with .1 mL of control peptide in the same buffer, using isoflurane anesthesia. All mice were harvested at 72 hours after removal of DSS and the colons were examined histologically.

Histologic Mucosal Injury Score Resected colons were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with H&E. The severity of mucosal injury was graded similarly to that described previously.35,37 The injury scale was graded from 0 to III, as follows: grade 0 ⫽ normal; grade I ⫽ distortion and/or destruction of the bottom third of glands and focal inflammatory infiltrate; grade II ⫽ erosions/destruction of all glands or the bottom two thirds of glands and inflammatory infiltrate with preserved surface epithelium; and grade III ⫽ loss of entire glands and surface epithelium. Specimens were examined without knowledge of the experimental group. The total number of low-power (10⫻) fields showing grade III colitis was determined for each specimen. An overall crypt damage score also was calculated by giving grade I, II, and III scores of 1, 2, and 3, respectively. Each low-power field was graded, and the percentage of each specimen with each score was calculated and added to give the final crypt damage score (range, 0 –3.00). For example, the same length of colon was examined for each specimen, and a specimen with 10% of fields with a score of 1, 25% of fields with a score of 2, and 25% of fields with a score of 3 would have a crypt damage score of (0.1)1 ⫹ (.25)2 ⫹ (.25)3 ⫽ 1.35.

Statistical Analysis The mean ⫾ standard error of the mean was calculated for variables in each experimental group and analyzed by analysis of variance (ANOVA) and the Student t test (2-tailed) or the Fisher exact test, as appropriate. A P value of less than .05 was considered significant. If a significant difference was found using an ANOVA, a post hoc t test was performed to reveal the source(s) of the significance.

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Results Design of Recombinant Muc3 Proteins

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We synthesized a series of recombinant GST fusion proteins corresponding to both mouse Muc3 EGF-like domains (m3EGF1,2), the first EGF-like domain (m3EGF1), or the second EGF-like domain (m3EGF2) in E coli and purified them using glutathione-agarose columns (Figure 1). Rat Muc3 has been shown to be cleaved posttranslationally between its 2 EGF-like domains. The resulting 2 subunits re-associate through a noncovalent bond that can be broken by 2% SDS and boiling.38,39 Our recombinant m3EGF1,2 appeared as a predominant single band in reducing Coomassie-stained gels at the expected molecular weight of 54 kilodaltons (Figure 1E). Treatment of recombinant m3EGF1,2 by boiling for 5 minutes in 2% SDS did not result in a change in molecular weight; indicating that this type of cleavage did not occur in our recombinant GST fusion protein (data not shown). Similarly, the recombinant m3EGF1 and the m3EGF2 appeared as single bands of 35.6 and 36.5 kilodaltons, respectively, on reducing Coomassie-stained gels (Figure 1E). Under nonreducing SDS polyacrylamide gel electrophoresis conditions, m3EGF1,2 and m3EGF2 appear as high molecular weight complexes (data not shown). To determine if all cysteines in the recombinant proteins were involved in disulfide bonds, the free thiol content of the proteins was determined. The thiol content was determined to be near zero in control peptides (mouse gastric mucin tandem repeat peptide and EGF), which are predicted to lack free thiols. The positive control peptide mouse gastric mucin nonrepeat peptide containing 2 free thiols was measured to contain 1.6 free cysteines per peptide (Table 1). GST alone also had negligible free thiols. m3EGF1,2 and m3EGF1 had very little measurable thiol, suggesting that all the cysteines were found in disulfide bonds. Interestingly, m3EGF2 appeared to have a free cysteine. Without extensive evaluation of the pairing of the cysteine residues in each of our proteins, we can only hypothesize as to the apparent single free cysteine in m3EGF2. The free cysteine measured in m3EGF2 could be the result of differences of the folding of this protein in solution, resulting in incomplete intermolecular pairing of a cysteine from one protein molecule with that of another protein molecule, leaving a single unpaired cysteine in each protein.

Table 1. Thiol Measurement in Recombinant Peptides

Peptide GST m3EGF1,2 m3EGF1 m3EGF2 EGF Mouse gastric mucin tandem repeat peptide Mouse gastric mucin nonrepetitive peptide (MGMnr)

Predicted no. Measured no. of of cysteines free cysteines in sequence per peptide 4 22 12 14 6 0

.05 .34 .12 1.37 .01 .00

2

1.57

NOTE. m3EGF1,2, m3EGF1, and m3EGF2 refer to the GST fusion proteins, and the predicted number of cysteines includes the cysteines in GST plus the cysteines in the indicated Muc3 domain.

Effect of Muc3 Recombinant Peptides on Cell Proliferation The effect on cell proliferation was determined in LoVo and A431 cells over 24 hours. As depicted in Figure 2A, treatment of LoVo cells with m3EGF1, m3EGF2, and m3EGF1,2 did not result in any significant changes in cell numbers after 24 hours. Similarly, there was no significant effect on cell numbers after treatment of A431 and YAMC cells with 1–50 ␮g/mL of m3EGF1,2 (Figure 2B and C).

Recombinant m3EGF1,2 Stimulates Cell Migration YAMC cells, human epithelial cell lines A431, and LoVo human colon cancer cells are known to contain ErbB receptors, therefore, we examined the effect of recombinant Muc3 EGF domain proteins on cell migration. YAMC cells treated with m3EGF1,2 showed significantly increased wound closure over 24 hours compared with no treatment (P ⬍ .05), whereas EGF-treated wounds attained complete wound closure. A dose response was shown with m3EGF1,2 (Figure 3A). Human A431 cells treated with 10 ␮g/mL m3EGF1,2 for 18 –24 hours showed a 215% increase in cell migration above controls (P ⬍ .05). In A431 cells recombinant EGF at 1 ng/mL stimulated cell migration to nearly 300% of controls (Figure 3B). In contrast, the truncated Muc3 cysteine-rich recombinant proteins m3EGF1 and m3EGF2 did not alter cell migration (Figure 3C). LoVo human colon cancer cells treated with 1 ␮g/mL of m3EGF1,2 showed a 2-fold increase in cell migration over 24 hours compared with controls, which was similar to the migration induced by 1 ng/mL recombinant EGF (Figure 3D, left panel). A combination of m3EGF1 (5 ␮g/mL) and m3EGF2 (5 ␮g/mL) did not show a significant increase in cell migration when compared with SF, m3EGF1 (10 ␮g/mL), or m3EGF2 (10 ␮g/ mL) controls (Figure 3D, right panel). A dose response was shown with a further 2.6-fold increase in cell migration with 10 ␮g/mL of m3EGF1,2. Subsequent increases in cell migration with doses of 20 ␮g/mL or more were not observed. A431 cells have been shown to contain high amounts of EGF receptor, and we observed greater amounts of cell migration in response to purified human EGF (Figure 4A and B) compared with LoVo cells; and the response of A431 to EGF was greatest at 24 hours of exposure compared with 18 –24 hours as in Figure 3C. Therefore, to determine if recombinant Muc3 EGF domain proteins act via stimulation of the EGF receptor, A431 cells were used with a longer period of incubation of 24 hours, with and without tyrphostin (AG1478), an inhibitor of this receptor. Tyrphostin (150 nmol/L) inhibited EGF-induced A431 cell migration, but not cell migration induced by m3EGF1,2 (Figure 4A). In contrast to the different migration response to purified human EGF noted in A431 and LoVo cells, the response of these 2 cell lines to m3EGF1,2 was uniform. To determine if tyrosine phosphorylation was required for m3EGF1,2-induced cell migration, A431 cells were pretreated with 55.5 ␮mol/L genistein. This resulted in significant inhibition of EGF-induced cell migration, and complete inhibition of cell migration induced by m3EGF1,2 (Figure 4B). LoVo cells contain endogenous MUC3A as shown by Williams et al3 and PCR and mRNA slot-blot analysis. To determine the involvement of endogenous MUC3A on cell migration, we attenuated MUC3A expression using siRNA. Figure 4C shows the decrease in MUC3A levels as measured by reverse-

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A LoVo 300 *

% Cell Proliferation

250 200 150 100 50

on tro ST l (1 0µ g/ m G L) ST (1 µg G /m ST L) (0 .1 µg G ST /m L) (0 .0 m 1µ 3E g/ m G F1 L) (5 0µ m 3E g/ m G L) F1 m (1 3E µg G / m F1 L) (0 m .1 3E µg G /m F1 L) (0 .0 m 1 3E µg /m G F2 L) (1 0µ m 3E g/ m G L) F2 m (1 3E µg G / m F2 L) m (0 3E .1 µg G F2 /m L) (0 m .0 3E 1µ G g F1 /m L) ,2 (5 m 0µ 3E g/ G m F1 L) ,2 m 3E (1 µg G F1 /m ,2 L) m (0 3E .1 G µ F1 g/ m ,2 L) (0 .0 1µ g/ m L) 10 % FB S

0

G

Treatment

B A431 * 150 100 50 0 Control

C

GST (10µg/mL)

m3EGF1,2 (10µg/mL)

m3EGF1,2 (20µg/mL)

m3EGF1,2 (50µg/mL)

EGF (1ng/mL)

YAMC 150

*

100

50

0 Control

GST m3EGF1,2 m3EGF1,2 m3EGF1,2 (10µg/mL) (10µg/mL) (5µg/mL) (1µg/mL)

transcription PCR analysis after transfection of LoVo cells with MUC3A-specific siRNA, nonspecific siRNA, or no siRNA. Quantification of MUC3A mRNA levels by slot-blot analysis confirmed the significant reduction in MUC3A mRNA resulting from MUC3A siRNA transfection compared with nonspecific siRNA transfection (Figure 4D). The basal level of cell migration in LoVo cells with diminished endogenous MUC3A was decreased significantly compared with untransfected cells and cells transfected with the nonspecific siRNA (Figure 4E).

Recombinant m3EGF1,2 Does Not Activate EGF Receptors To analyze further whether m3EGF1,2 caused activation or phosphorylation of the EGF (ErbB1) receptor, A431 cells were treated with recombinant proteins and cell lysates were examined for phosphotyrosine content. The EGF receptor also was immunoprecipitated and analyzed by immunoblot using an antiphosphotyrosine antibody to assess EGF-receptor

EGF

phosphorylation. Treatment of cells with recombinant EGF at 1 ng/mL for 1, 5, 10, 30, and 60 minutes resulted in a significant increase in phosphotyrosine at 175 kilodaltons compared with control treatments (Figure 5A). In contrast, similar levels of baseline 175-kilodalton phosphotyrosine reactivity was observed in A431 cells in serum-free medium or treated with m3EGF1,2 or GST at these time points. Also illustrated in Figure 5A is an increase in phosphotyrosine bands between 80 and 120 kilodaltons that was observed in cell lysates from A431 cells treated with m3EGF1,2 at the 10-minute time point. This was repeated twice with the same results. The characterization of intracellular signaling pathways and proteins phosphorylated in response to m3EGF1,2 will require further studies. Further analysis of EGF or ErbB-receptor binding was confirmed by immunoprecipitation followed by phosphotyrosine blotting in cells treated at selected time points with recombinant EGF or m3EGF1,2. Triplicate experiments showed a significant increase in EGF receptor phosphoryla-

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% cell proliferation

200

% Cell Proliferation

Effect of recombinant Muc3 peptides on proliferation. (A) Proliferation of LoVo colon cancer cells as measured by dimethylthiazole diphenyltetrazolium bromide after 24 hours. For a positive control, cells were grown in 10% fetal bovine serum (FBS). No significant differences (NS) were observed in cell numbers measured by dimethylthiazole diphenyltetrazolium bromide after treatment of cells with serum-free (SF) control, GST, m3EGF1, m3EGF2, or m3EGF1,2 (ANOVA, P ⬍ .001). Student t test, *P ⬍ .05 vs control, n ⫽ 6 –12. (B) Effect of recombinant GST peptide, m3EGF1,2 and recombinant EGF on A431 cell numbers after 24 hours, expressed as percentage of cell numbers in serum-free control medium (ANOVA, P ⬍ .001). Student t test, *P ⬍ .05 vs control. (C) Effect of recombinant GST peptide, m3EGF1,2, and recombinant EGF on YAMC cell numbers after 24 hours, expressed as percentage of control cell numbers in control medium (ANOVA, P ⬍ .05). *P ⬍ .001 vs control.

C

Figure 2.

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Inhibitor studies of cell migration. (A) A431 cell migration over 24 hours in response to m3EGF1,2 (10 ␮g/mL) or EGF (1 ng/mL) with and without the specific EGF/ErbB1-receptor inhibitor tyrphostin (TYR), (AG1478) (150 nM). SF, serum-free medium (ANOVA, P ⬍ .001). *P ⬍ .05 vs SF and GST controls, n ⫽ 6 wells for each condition. (B) A431 cell migration over 24 hours in response to m3EGF1,2 10 ␮g/mL or EGF 1 ng/mL with and without a general inhibitor of tyrosine phosphorylation, genistein (GEN) (55.5 ␮mol/L) (ANOVA, P ⬍ .001). ⴱP ⬍ .04 vs SF and GST controls. ⫹P ⬍ .02 vs EGF, n ⫽ 6 each treatment. (C) MUC3A and actin mRNA levels in LoVo cells after a 48-hour treatment with MUC3A siRNA or nonspecific siRNA. RNA was isolated from untransfected LoVo cells or cells transfected with MUC3A siRNA or nonspecific siRNA, reversed transcribed, and amplified by PCR using MUC3A or actin primers. The PCR products were separated on an agarose gel and visualized by ethidium bromide staining. Representative samples are shown. Lane 1, untransfected; lane 2, transfected with nonspecific siRNA; and lane 3, transfected with MUC3A siRNA. PCR products were quantitated by densitometric analysis of the ethidium bromide–stained gel (n ⫽ 3 replicates). P ⬍ .01 for MUC3 siRNA vs no siRNA or nonspecific siRNA. (D) MUC3A-RNA levels quantitated by slot blot. RNA from LoVo cells transfected with MUC3A siRNA or nonspecific siRNA was blotted on a nylon membrane and probed with a radiolabeled MUC3A or actin cDNA probe. The membrane was exposed to radiographic film and bands were quantitated by densitometry. A representative experiment is shown in the graph. *P ⫽ .05, n ⫽ 3 replicates. (E) Percentage of LoVo cells migrating over 24 hours in response to no siRNA, MUC3A siRNA, or nonspecific control siRNA in SF media (ANOVA, P ⬍ .03). **P ⬍ .03 vs no siRNA and control siRNA in SF media, n ⫽ 6 replicates.

Figure 4.

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Figure 3.

Effect of recombinant Muc3 peptides on cell migration. (A) Wounds were made in YAMC cell monolayers using a spinning circular disc and the percentage of total wound closure measured at 24 hours. YAMC cells treated with .05–10 ␮g/mL m3EGF1,2 show increased cell migration above control with no treatment (*P ⬍ .05), and a dose response is shown. EGF (1 ng/mL) was used as a positive control and resulted in the attainment of 100% wound closure at 24 hours (ANOVA, P ⬍ .03). *P ⬍ .05 vs no treatment, n ⫽ 5 each condition. (B) Photomicrographs of A431 cell migration after 24 hours in serum-free medium, medium with GST control protein (10 ␮g/mL), medium with m3EGF1,2 (10 ␮g/mL), and medium with recombinant EGF (1 ng/mL) (200⫻). In these wells baseline migration over the indicated scrape lines was observed in cells in serum-free medium and treated with GST control. (C) A431 cell migration in response to m3EGF1,2, m3EGF1, m3EGF2 over 18 –24 hours represented as a percentage of baseline cell migration in serum-free medium normalized to 100% (percentage of cell migration) (ANOVA, P ⬍ .002). *P ⬍ .05 for m3EGF1,2 vs control (GST); EGF vs SF; n ⫽ 6 wells for each condition. (D) Migration of LoVo cells treated with varying concentrations of proteins represented as the percentage of baseline control cell migration after 24 hours (percentage of cell migration) (ANOVA, P ⬍ .03, left panel; ANOVA, P ⬍ .002, right panel). *P ⬍ .05 vs SF, n ⫽ 6 wells for each condition.

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Figure 5.

Recombinant Muc3 peptide and EGF-family receptor phosphorylation. (A) Cell lysates from A431 cells treated with serum-free medium (SF) (lane 1), EGF (1 ng/mL) (lane 2), GST (10 ␮g/mL) (lane 3), or m3EGF1,2 (10 ␮g/mL) (lane 4), for 1, 5, 10, 30, or 60 minutes were immunoblotted with antiphosphotyrosine antibody (ANTI-PY) and with anti–EGF-receptor antibody. Treatment with recombinant EGF resulted in a significant increase in phosphotyrosine reactivity at 175 kilodaltons, whereas treatment with serum-free medium, control GST, and m3EGF1,2 showed baseline levels of 175 kilodalton phosphotyrosine reactivity. Note that at 10 minutes an increase in phosphotyrosine bands between 80 and 120 kilodaltons was observed in cells treated with m3EGF1,2. (B) Representative blot from 1 experiment of A431 cells exposed to EGF (1 ng/mL), serum-free media (SF), m3EGF1,2 (10 ␮g/mL), or GST (10 ␮g/mL) for 60 minutes. Lysates were immunoprecipitated with anti–EGF receptor, and then were immunoblotted with antiphosphotyrosine or with anti–EGF receptor. (C) Densitometric quantification in arbitrary densitometric units (y-axis) of phosphotyrosine reactivity of immunoprecipitated EGF receptor at 60 minutes from 3 separate experiments. EGF (10 ng/mL) caused a significant increase in tyrosine phosphorylation compared with SF, GST (10 ␮g/mL), and m3EGF1,2 (10 ␮g/mL). *P ⬍ .05 vs SF and GST controls. n ⫽ 3 separate experiments. (D) YAMC cells were exposed to EGF (1 ng/mL) for 5 minutes or serum-free media (SF), m3EGF1,2 (10 ␮g/mL), or GST (10 ␮g/mL) for 30 minutes. Lysates were immunoprecipitated with anti-EGF receptor, anti-ErbB2, or anti-ErbB3, which then were immunoblotted with antiphosphotyrosine or with anti–EGF receptor, anti–ErbB2 receptor, or anti–ErbB3 receptor, respectively.

tion by recombinant EGF, but not by m3EGF1,2 or control peptide at 60 minutes (Figure 5B and C). Subconfluent cultures of YAMC cells similarly were treated with 10 ␮g/mL of m3EGF1,2 and a similar concentration of GST for 30 min-

utes, or with 1 ng/mL recombinant EGF for 5 minutes. Cell lysates were immunoprecipitated with antibodies to EGF receptor, ErbB2, and ErbB3. Phosphorylation of EGF receptor and ErbB2 occurred in response to EGF, however,

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Figure 6. Effect of Muc3 and MUC3A expression on apoptosis. (A) Western blotting using anti-Flag antibody. Lanes: stably transfected LoVo clone LhM3c14 cytoplasmic fraction (LhM3c14 cyt) and membrane fraction (LHM3C14 MEM), mock-transfected LoVo cell clone membrane fraction (LMOCK MEM), nontransfected LoVo membrane fraction (MEM). (B) Percentage change in apoptosis with (⫹) or without (⫺) TNF-␣ (100 ng/mL) treatment for 48 hours. Cell lines included parental LoVo, LhM3c14, Lmock, and nontransfected LoVo cells pretreated with m3EGF1,2 (10 ␮g/mL) or GST (5 ␮g/mL) for 1 hour before addition of TNF-␣. Baseline apoptosis ⫽ 3.9%. *P ⬍ .016 vs LoVo (⫹), ^P ⬍ .009 vs GST, ⫹P ⬍ .002 vs Lmock; n ⫽ 2–3 plates/treatment and 3 fields counted/plate. (C) Percentage change in apoptosis in nontransfected LoVo cells with (⫹) or without (⫺) sequential interferon-␥ and anti-Fas antibody treatment for 72 hours. Cells were pretreated with m3EGF1,2 (10 ␮g/mL), m3EGF1 (10 ␮g/mL), m3EGF2 (10 ␮g/mL), GST (10 ␮g/mL), EGF (10 ng/mL), or m3EGF1 (5 ␮g/mL) ⫹ m3EGF2 (5 ␮g/mL) before addition of anti-Fas antibody. Baseline apoptosis ⫽ 1.1% and .9% for left and right panels, respectively (ANOVA, P ⬍ .001, within both panels). *P ⬍ .005 vs LoVo, LoVo ⫹ m3EGF1, m3EGF2 and GST controls (left panel). *P ⬍ .004 vs LoVo and LoVo ⫹ m3EGF1 ⫹ m3EGF2 (right panel); n ⫽ 2 plates/treatment and 2 fields counted/plate. (D) Percentage change in apoptosis with (⫹) or without (⫺) sequential interferon-␥ and anti-Fas antibody treatment for 48 hours. Cell lines included LhM3c14 and Lmock (ANOVA, P ⬍ .001). Baseline apoptosis ⫽ 2.3%. *P ⬍ .025 vs Lmock (⫺) and LhMc14 (⫹) n ⫽ 3 plates/treatment and 3 fields counted/plate. (E) LoVo cells were treated with increasing concentrations of the EGF-receptor inhibitor, tyrphostin (AG1478). This resulted in a dose-dependent reversal of the anti-apoptosis effect of recombinant EGF, but did not affect the anti-apoptosis effect of m3EGF1,2 (ANOVA, P ⬍ .001). Baseline apoptosis ⫽ 2.0%. *P ⬍ .03 vs m3EGF1,2 (same tyrphostin concentrations). (Note that maximal apoptosis observed varied from experiment to experiment because of the limited half-life of the activity of the anti-Fas in storage).

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Figure 7.

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Figure 8.

Effect of recombinant Muc3 peptides on DSS-induced colitis. Mice were treated with 5% DSS in drinking water for 7 days, followed by treatment with m3EGF1,2 (100 ␮g), GST (100 ␮g), or BSA (100 ␮g) per rectum at 24 and 48 hours, followed by colon examination at 72 hours. Crypt damage scores and mean number of fields/specimen with grade III ulceration from the (A and B) middle to distal mouse colons and the (C and D) proximal colons are represented. (A) ANOVA P ⬍ .005. *P ⬍ .001 vs GST; P ⫽ .07 vs PBS. (B) ANOVA, P ⬍ .009; *P ⬍ .005 vs GST; *P ⫽ .05 vs PBS. (C and D) ANOVA, P ⫽ NS.

m3EGF1,2 treatment did not result in phosphorylation of EGF receptor, ErbB2, or ErbB3 (Figure 5D).

Endogenous MUC3A and Exogenous Muc3 Peptides Inhibit Apoptosis A human MUC3A transmembrane-EGF1,2 domain construct was transfected stably into LoVo human colon cancer cells (Figure 1D). LoVo cell clone LhM3c14 expressed high levels of flag-tagged human MUC3A EGF1,2 in the cell membrane fractions; this was absent from LhM3c14 cytoplasmic fractions,

Lmock, and parental LoVo cells (Figure 6A). Apoptosis was induced in parental LoVo human colon cells, Lmock cells, and LhM3c14 cells using TNF-␣. The stable transfectant clone LhM3c14 was markedly resistant to TNF-␣–induced apoptosis (Figure 6B). Similarly, pretreatment of parental LoVo cells with 10 ␮g/mL m3EGF1,2 reduced TNF-␣–induced apoptosis, whereas pretreatment with GST did not (Figure 6B). Apoptosis induced by sequential interferon-␥ and anti-Fas antibody treatment was reduced markedly in parental LoVo cells by pretreatment with m3EGF1,2 or EGF control compared with pretreat-

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Figure 7.

Effect of recombinant Muc3 peptides on acetic acid–induced colitis. (A) Crypt damage score (CDS) at 30 hours after acetic acid administration in mice that received treatment with m3EGF1,2 (100 ␮g) or BSA as a control (100 ␮g) in PBS per rectum at 12 and 24 hours after acetic acid. *P ⬍ .05 vs control; n ⫽ 10 mice/treatment. (B) Mean number of low-power (10⫻) fields per specimen with complete grade III ulceration at 30 hours after acetic acid administration in mice treated twice with 100 ␮g m3EGF1,2 or control peptide 100 ␮g BSA in PBS. *P ⬍ .03 vs control; n ⫽ 10 mice/treatment. (C) CDS at 30 hours after acetic acid administration in mice that received treatment with GST, m3EGF1, m3EGF2, or m3EGF1,2 per rectum at 12 and 24 hours after acetic acid. *P ⬍ .036 vs GST, *P ⬍ .05 vs m3EGF2 (ANOVA, P ⬍ .05). (D) Mean number of low-power (10⫻) fields per specimen with complete grade III ulceration at 30 hours after acetic acid administration in mice that received treatment with GST, m3EGF1, m3EGF2, or m3EGF1,2 per rectum at 12 and 24 hours after acetic acid. *P ⬍ .03 vs m3EGF2. (E and F) Distal mouse colon 30 hours after induction of acetic acid colitis and treatment with 100 ␮g m3EGF1,2, showing (E) normal colon crypts or grade 0 crypt damage and (F) grade 1 crypt damage with shortening of the crypts and focal inflammatory cell infiltrate. (G and H) Distal mouse colon 30 hours after induction of acetic acid colitis and treatment with 100 ␮g BSA in PBS showing (G) grade II crypt damage with partial loss of glands and preserved surface epithelium, and (H) grade III crypt damage with destruction and loss of glands and surface epithelium. H&E magnification, 200⫻.

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Figure 9. Effect of recombinant Muc3 protein on acetic acid–induced apoptosis. (A) Mean number of apoptotic cells determined by TUNEL assay in the distal colon of mice 30 hours after acetic acid administration in mice treated twice with 100 ␮g m3EGF1,2 or control peptide 100 ␮g BSA in PBS (n ⫽ 10 mice each treatment). (B) Representative distal colon 30 hours after acetic acid and control enema treatments. TUNEL-positive nuclei are stained darkly. (C) Representative distal colon 30 hours after acetic acid and m3EGF1,2 enema treatments (magnification, 200⫻).

ment with truncated Muc3 peptides m3EGF1, m3EGF2, a combination of m3EGF1 and m3EGF2, or with control GST peptide (Figure 6C). The stable transfectant clone LhM3c14 showed significantly less apoptosis compared with the mock transfectant Lmock in response to sequential interferon-␥ and anti-Fas antibody treatment (Figure 6D). Note that the maximal apoptosis observed varied from experiment to experiment because of the limited half-life of the activity of the anti-Fas in storage, despite avoiding any freeze-thaw. To verify that the anti-apoptosis effect of the m3EGF1,2 protein was not caused by activation of the EGF receptor, cells were treated with increasing concentrations of the EGF-receptor inhibitor, tyrphostin (AG1478). This resulted in a dosedependent reversal of the anti-apoptosis effect of recombinant EGF, but did not affect the anti-apoptosis effect of m3EGF1,2 (Figure 6E).

Recombinant m3EGF1,2 Accelerates Healing of Experimental Colitis To determine if our recombinant Muc3 peptides could influence the healing or regeneration of intestinal mucosa, 2 different mouse models of acute colitis were used. In the first model, acute colonic injury was induced in mice by 5% acetic acid enemas, followed by the administration of recombinant protein or control enemas at 12 and 24 hours. The animals were killed at 30 hours to determine the extent of mucosal damage. Treatment of mice with 100 ␮g of m3EGF1,2 per rectum at 12 and 24 hours after acetic acid reduced the total crypt damage score by 45% compared with enemas containing 100 ␮g bovine serum albumin (BSA) in PBS buffer (P ⫽ .05) (Figure 7A). This was largely owing to the significant reduction in total or grade III mucosal ulcerations from 8.2 ⫾ 1.6 low-power fields/specimen in control-treated mice to 3.5 ⫾ 1.4 low-power fields/ specimen in mice treated with 100 ␮g m3EGF1,2 peptide enemas (P ⫽ .038) (Figure 7B). Figure 7E–H illustrates the

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histologic difference between normal mouse colonic mucosa and grade I, II, and III damage. The experiment was repeated using control enemas containing PBS buffer with 100 ␮g of recombinant GST, compared with enemas containing 1 ␮g, 50 ␮g, or 100 ␮g of recombinant m3EGF1,2; 100 ␮g m3EGF1; and 100 ␮g m3EGF2. Mice treated at 12 and 24 hours with enemas containing 100 ␮g of m3EGF1,2 showed a significant 62% reduction in crypt damage score (Figure 7C) and a 79% reduction in grade III mucosal ulceration (Figure 7D) compared with mice treated with enemas containing 100 ␮g GST control protein. Mice treated with enemas containing 1 ␮g m3EGF1,2 and 50 ␮g m3EGF1,2 had nonsignificant reductions of 29%– 40% in crypt damage scores and 38%– 40% in grade III ulceration compared with control enema treatment. In contrast, enemas containing 100 ␮g m3EGF1 or 100 ␮g m3EGF2 had no effect on crypt damage score or total mucosal ulceration (Figure 7C and D). Administration of 5% DSS in drinking water results in an acute colitis that predominates in the distal colon and heals with withdrawal of the DSS. Mice treated with 100 ␮g m3EGF1,2 per rectum at 12 and 24 hours after DSS withdrawal and examined at 72 hours after DSS withdrawal showed a 38% reduction in crypt damage scores in the distal colon compared with mice treated with control enemas with GST or BSA (P ⬍ .005) (Figure 8A). This was primarily owing to a 53% decrease in the mean number of fields/ specimen with total grade III mucosal ulceration; from a mean of 8.5 ⫾ 1.1 fields/specimen in all controls to 4.0 ⫾ .8 fields/specimen in mice treated with m3EGF1,2 (P ⬍ .005) (Figure 8B). Mucosal damage was less in the proximal colon, and no significant differences were observed in crypt damage scores or in the number of fields with grade III ulceration in treated and control mice (Figure 8C and D). To determine if a similar reduction in apoptosis was observed in vivo, specimens from mice with acetic acid colitis were studied using a TUNEL assay to detect apoptotic cells. The number of apoptotic cells in the epithelial layer of the distal colon were reduced markedly in mice treated with m3EGF1,2 enemas compared with control-treated mice (Figure 9). These specimens also were assayed for a neutrophil marker, myeloperoxidase, determined by immunohistochemistry. The myeloperoxidase assay failed to show any difference between the control and m3EGF1,2-treated mice (data not shown), likely owing to the lack of sensitivity of the assay used. Further studies of changes in inflammatory mediators that accompany m3EGF1,2 treatment are planned.

Discussion These data indicate that the extracellular cysteine-rich domains of the Muc3 mucin stimulate cell migration in vitro and accelerate wound healing in vivo. These effects are found within a relatively narrow dose range and were observed only with the Muc3 recombinant mucin that included both the EGF1 and EGF2 cysteine-rich domains. The mechanism of enhanced cell migration appears to require tyrosine phosphorylation, but does not appear to be via direct activation of EGF receptor (ErbB1), ErbB2, or ErbB3. Both exogenous m3EGF1,2 peptide and the endogenous expression of transfected human MUC3A transmembrane-EGF1,2 in colon cells resulted in reduced apoptosis induced by either TNF-␣ or Fas-receptor stimulation. These data suggest a potential physiologic role for membrane-bound mucins in mucosal restitution by the stimu-

lation of cell migration and inhibition of apoptosis; and also suggest a role for mucin cysteine-rich recombinant proteins as therapeutic agents for intestinal wound healing. Mucin preparations, both alone and in combination with trefoil proteins, have been shown previously to promote restitution of wounded epithelium.40 However, the mucin preparations used were heterogeneous and likely contained more than one mucin species and other interacting proteins, preventing the assignment of a wound-healing role to a specific mucin. Trefoil Factor Family (TFF) proteins are small (7–12 kilodaltons) cysteine-rich proteins that are secreted with mucins in the gastrointestinal tract. Trefoil proteins have been shown to stimulate cell migration in intestinal cells, to protect intestinal epithelial cell monolayers against injurious agents, and to accelerate healing of experimental models of inflammatory bowel disease or ulcers.41 The mechanism of this protection is not completely known and no cell-surface receptor for trefoils has been identified to date. Trefoil peptides can trigger various signaling cascades, involving extracellular regulated kinase/ ERK1,2, protein kinase B/Akt, and transactivation of EGF receptor in certain cell lines.41 The data presented here indicate that the Muc3 mucin cysteine-rich peptide m3EGF1,2 may cause tyrosine phosphorylation, but further studies to determine the specific signaling pathways affected are in progress. Little information exists regarding the biochemical characteristics of intestinal membrane-bound mucins, and there are no specific reagents, antibodies, or purification techniques that would easily allow for quantification of endogenous membranebound mucins.42 The concentrations of recombinant m3EGF1,2 used in the present studies likely represent pharmacologic concentrations of these proteins rather than physiologic concentrations. To determine if endogenous MUC3A mucin may have a similar effect on cell migration, siRNA was used to knock down endogenous MUC3A mRNA in LoVo cells. This resulted in inhibition of cell migration, suggesting that the effects of recombinant m3EGF1,2 may reflect biologic effects of endogenous MUC3A. Further studies such as these, in addition to transgenic and gene deletion studies in vivo, are necessary to determine fully the role that endogenous membrane-bound mucins play in normal intestinal homeostasis. Both exogenous m3EGF1,2 and the stable expression of MUC3A EGF1,2 in LoVo cells inhibited apoptosis induced by TNF-␣ or anti-Fas antibody. Several other membrane-bound mucins and trefoils have shown anti-apoptotic properties in experimental systems. Transfection of the full-length MUC1 mucin in rat fibroblast cells resulted in reduced apoptosis in response to 1-〉-D-arabinofuranosylcytosine and gemcitabine. This was associated with involvement of both a phosphatidylinositol 3-kinase–mediated pathway and also with an increase in the Bcl-x anti-apoptotic protein by a phosphatidylinositol 3-kinase–independent pathway.43 Transfection of the MUC1 cytoplasmic domain alone also has been shown to inhibit apoptosis induced by hydrogen peroxide.44 Transfection of Muc4 in A375 melanoma cells results in reduced apoptosis in response to serum starvation.45 Exogenous administration of intestinal trefoil factor (TFF3) and expression of transfected TFF3 resulted in resistance to apoptosis in colon cell lines. This effect could be abrogated by inhibitors of phosphatidylinositol 3-kinase and EGF-receptor activation, indicating that the antiapoptotic effect of TFF3 may be mediated by involvement of these pathways.46 Increased cell migration and anti-apoptosis

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often are associated and share some common pathways, such as the phosphatidylinositol 3-kinase pathway.47– 49 These studies suggest that variable mechanisms may be involved in antiapoptotic effects induced by MUC1, Muc4, and trefoil factors; and suggest that protection from apoptosis by potentially diverse mechanisms is a property of peptides located or released at epithelial cell surfaces to promote epithelial cell survival and wound restitution. The specific cell migration and anti-apoptosis signaling pathways stimulated by m3EGF1,2 is the subject of ongoing research. MUC4 is expressed in a variety of simple and stratified epithelial tissues, including the respiratory tract, cervix, uterus, mammary gland, colon, cornea, conjunctiva, vagina, and oral tissues. In normal cells MUC4 is thought to function as a protective and anti-adhesive barrier, and in addition may have a role in receptor binding and cell signaling in response to external stimuli or damage. In cancer cells the large cell surface Muc4 protein has been shown to disrupt cell– cell adhesion and promote tumor growth and metastases.50,51 The Muc4 ASGP-2 extracellular domain contains 2 EGF-like regions (EGF1, EGF2) and has been shown to bind the cell surface transmembrane receptor ErbB2 via the EGF1 domain when both are expressed in the same cell. Thus, the Muc4 EGF1 domain is considered to be a novel intramembrane ligand for the ErbB2/HER2/Neureceptor tyrosine kinase, which can trigger a specific phosphorylation of ErbB2 in the absence of other ErbB ligands.52,53 However, the specific consequences of the interaction of the MUC4 EGF1 domain and the ErbB2 receptor in normal and cancer cells has not been elucidated completely. Currently, it is not known if the EGF domain(s) of soluble forms of Muc4 can act as a ligand for ErbB2 or other receptors.6 We could not show an association between m3EGF1,2 and ErbB1 or ErbB2 in the transfected LoVo LhM3c14 cell clone, possibly owing to the low levels of these receptors in this cell line (data not shown). Studies are in progress to co-express Muc3 EGF1,2 domains in cells with high-level ErbB receptors to determine if Muc3 functions as an intramembrane ligand for ErbB receptors in a similar fashion as Muc4. Exogenous administration of the m3EGF1,2 recombinant peptide resulted in enhanced mucosal regeneration in 2 mouse models of colitis. Acetic acid results in colitis through direct mucosal damage and loss of epithelium. DSS is a negatively charged sulfate polysaccharide that is thought to induce colitis by early changes in the hemodynamics of colonic circulation and as a result of direct injury of colonic epithelial cells and tight junctions.54,55 The improvement in histologic colitis in m3EGF1,2-treated mice was evident primarily by the reduction in the length of colon with complete denudation or grade III ulceration of the mucosa in treated animals, rather than to a reduction in lesser levels of grade I and II crypt destruction and inflammation. This may be owing to enhanced cell migration that allows for sealing the epithelial defect from the lumen, thus allowing for faster crypt regeneration. A reduction in the amount of apoptosis observed in the m3EGF1,2-treated colons may have allowed for this enhanced migration. Interestingly, the smaller recombinant m3EGF1 domain peptide and the m3EGF2 domain peptide, either alone or present together, did not promote cell migration or inhibit apoptosis in vitro, or accelerate mucosal regeneration in vivo. This suggests that the linker region in the m3EGF1,2 peptide may be important in maintaining the cysteine-rich. EGF-like domains in a configu-

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ration that is biologically active Further structural studies are needed to determine if the linker region itself, or if various combinations including the removal or truncation of the linker region with or without including an EGF-like domain, has an affect on the observed biologic activity. In addition, further studies are needed to synthesize m3EGF1,2 in other expression systems to determine if glycosylation and other factors affect its biologic activity. Restitution of the surface mucosal layer is the initial step in repair of mucosal injury, can occur over broad areas of damage within hours, and is independent of proliferation.56 Strategies to promote epithelial restitution may represent novel modalities to treat gastrointestinal injury. Topical application of recombinant SP trefoil (TFF2) and intestinal trefoil factor (TFF3) has been shown to reduce ethanol and indomethacin-induced gastric injury in mice.57 This effect in part may be mediated by cross-activation of EGF receptors because both trefoil factors TFF2 and TFF3 have been shown to induce phosphorylation of the EGF receptor in gastric and colon cancer cell lines.58,59 Enhanced epithelial restitution also has been used in human beings to treat ulcerative colitis. A synthetic preparation of a recombinant human epidermal growth factor protein has been shown in one human study to accelerate healing of ulcerative colitis.60 Stimulation of EGF receptors in colon epithelial cells influences multiple pathways related to proliferation, cell migration, adhesion, and prevention of apoptosis. EGF receptors are found in intestinal neoplasms; hence there is a concern that EGF peptides, and potentially trefoil peptides, may directly stimulate tumor cell growth or indirectly increase the chance of neoplastic transformation of regenerating colonic epithelial cells. In part because of this, currently the use of EGF in human beings has been limited to research protocols, and there are no other specific treatments that are used in human beings that target intestinal restitution. Although the exact mechanism of action remains to be determined, the Muc3 cysteine-rich EGF1,2 peptide appears to differ from EGF peptides and perhaps trefoil peptides by accelerating epithelial restitution without stimulating ErbB1-3, and hence may have a therapeutic advantage. Further studies related to the pharmacologic and therapeutic potential of membrane-bound mucin cysteine-rich domains in healing gastrointestinal mucosal injury are warranted. References 1. Ho SB, Shekels LL. Mucin and goblet cell function. In: Koch TR, ed. Colonic diseases. Totowa, NJ: Humana Press, 2004:53–72. 2. Hollingsworth MA, Swanson BJ. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer 2004;4:45– 60. 3. Williams SJ, Munster DJ, Quin RJ, Gotley DC, McGuckin MA. The MUC3 gene encodes a transmembrane mucin and is alternatively spliced. Biochem Biophys Res Commun 1999;261:83– 89. 4. Crawley SC, Gum JR, Hicks JW, Pratt WS, Aubert JP, Swallow DM, Kim YS. 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 –736. 5. Moniaux N, Escande F, Batra SK, Porchet N, Laine A, Aubert JP. Alternative splicing generates a family of putative secreted and membrane-associated MUC4 mucins. Eur J Biochem 2000;267: 4536 – 4544. 6. Komatsu M, Arango ME, Carraway KL. Synthesis and secretion of Muc4/sialomucin complex: implication of intracellular proteolysis. Biochem J 2002;368:41– 48.

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Received June 20, 2005. Accepted July 19, 2006. Address requests for reprints to: Samuel B. Ho, MD, Gastroenterology Section 111-D, Veterans Affairs San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, California 92161. e-mail: [email protected]; fax: (858) 552-4327. Supported by a VA Merit Review Award (S.B.H.), grant DK54993 from the National Institutes of Health (D.B.P.), and the Research Service of the Department of Veterans Affairs.

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