1. Volume 131, Issue 5, Pages 1501-1517 (November 2006)
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, Laurie L. Shekels 
Gastroenterology 
Volume 131, Issue 5, Pages (November 2006)
DOI: /j.gastro
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2. 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) Co-omassie-stained SDS polyacrylamide gel electrophoresis of purified GST-labeled proteins. Lanes: m3EGF1,2, m3EGF1, m3EGF2, and GST.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
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.
Gastroenterology  , DOI: ( /j.gastro )
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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 μg/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 μg/mL), medium with m3EGF1,2 (10 μg/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.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

5. Figure 4
Inhibitor studies of cell migration. (A) A431 cell migration over 24 hours in response to m3EGF1,2 (10 μg/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 μg/mL or EGF 1 ng/mL with and without a general inhibitor of tyrosine phosphorylation, genistein (Gen) (55.5 μmol/L) (ANOVA, P < .001). *P < .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.
Gastroenterology  , DOI: ( /j.gastro )
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6. 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 μg/mL) (lane 3), or m3EGF1,2 (10 μg/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 μg/mL), or GST (10 μg/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 μg/mL), and m3EGF1,2 (10 μg/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 μg/mL), or GST (10 μg/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.
Gastroenterology  , DOI: ( /j.gastro )
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7. 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-α (100 ng/mL) treatment for 48 hours. Cell lines included parental LoVo, LhM3c14, Lmock, and nontransfected LoVo cells pretreated with m3EGF1,2 (10 μg/mL) or GST (5 μg/mL) for 1 hour before addition of TNF-α. 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-γ and anti-Fas antibody treatment for 72 hours. Cells were pretreated with m3EGF1,2 (10 μg/mL), m3EGF1 (10 μg/mL), m3EGF2 (10 μg/mL), GST (10 μg/mL), EGF (10 ng/mL), or m3EGF1 (5 μg/mL) + m3EGF2 (5 μg/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-γ 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).
Gastroenterology  , DOI: ( /j.gastro )
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8. 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 μg) or BSA as a control (100 μg) 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 μg m3EGF1,2 or control peptide 100 μg 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 μg 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 μg 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×.
Gastroenterology  , DOI: ( /j.gastro )
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9. 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 μg), GST (100 μg), or BSA (100 μg) 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 < *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.
Gastroenterology  , DOI: ( /j.gastro )
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10. 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 μg m3EGF1,2 or control peptide 100 μg 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×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions