1. Volume 132, Issue 5, Pages 1877-1889 (May 2007)
Matrix Metalloproteinase-9 Regulates MUC-2 Expression Through Its Effect on Goblet Cell Differentiation 
Pallavi Garg, Anupama Ravi, Neal R. Patel, Jesse Roman, Andrew T. Gewirtz, Didier Merlin, Shanthi V. Sitaraman 
Gastroenterology 
Volume 132, Issue 5, Pages (May 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
MMP-9−/− mice exhibit up-regulation of goblet cells. (A) WT and MMP-9−/− mice colonic tissues were harvested and fixed with paraffin. The sections were stained with AB pH 2.5 and PAS stains (original magnification, ×10 and ×40). Photomicrographs shown here are representative sections from 8 animals per group. (B) WT and MMP-9−/− mice colonic tissues were harvested and fixed with paraffin. The sections were stained with AB, pH 2.5, and PAS stains. Goblet cells in the crypts of the proximal colon were counted. Four to 6 fields per section with 2 individual sections from each animal were counted, and goblet cells/crypt are represented as mean ± SE (n = 5), significantly different from WT (P < .01) (C) Western blot analysis of carbonic anhydrase-I (CA-I) expression in WT and MMP-9−/− mice was done by homogenizing colon samples in cell lysis buffer. Equal amounts of protein were separated by SDS-PAGE and probed with CA-I antibody as described in the Materials and Methods section. β-actin served as the loading control. Each lane shows protein (30 μg/lane) from an individual mouse.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
MUC-2 is up-regulated in MMP-9−/− mice. (A) RNA was extracted by homogenizing tissues from wild-type and MMP-9−/− mice colons, and RT-PCR was performed using MUC-2-specific primers as described in the Materials and Methods section. MUC-2/18S RNA was quantitated by scanning densitometry and represented as percentage change of MMP-9−/− mice compared with WT mice. Values are representative of 3 individual experiments, mean ± SE; n = 5 for WT and MMP-9−/− mice. (B) Western blot analysis of MUC-2 expression in WT and MMP-9−/− mice was done by homogenizing colon samples in cell lysis buffer. Equal amounts of protein were separated by SDS-PAGE and probed with MUC-2 antibody as described in the Materials and Methods section. β-actin served as the loading control. Each lane shows protein (30 μg/lane) from an individual mouse. Western blot was quantitated by scanning densitometry and represented as percentage change of MUC-2/β-actin in MMP-9−/− mice compared with WT mice. Values are representative of 3 individual experiments, mean ± SE; n = 5 for WT and MMP-9−/− mice. (C) WT and MMP-9−/− mice colon tissues were harvested and fixed with paraffin, processed for confocal microscopy using anti-MUC-2 (green), and counterstained with rhodamine phalloidin (red). Confocal microscopy was performed using Zeiss LSM microscope (original magnification, 40×). Photomicrographs shown here are representative sections from 5 animals per group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
MMP-9−/− show altered goblet cell differentiation. (A) Western blot analysis of NICD, Notch-1, and KLF-4 expression in WT and MMP-9−/− mice was done by homogenizing colon samples in cell lysis buffer. Equal amounts of protein were separated by SDS-PAGE and probed with NICD, Notch-1, and KLF-4 antibodies as described in the Materials and Methods section. β-actin served as the loading control. Each lane shows protein (30 μg/lane) from an individual mouse and is representative of Western blots done on 5 mice in each group. Western blot was quantitated by scanning densitometry and represented as percentage change of Notch-1, NICD, or KLF-4/β-actin in MMP-9−/− mice compared with WT mice. Values are representative of 3 individual experiments, mean ± SE; n = 5 for WT and MMP-9−/− mice. (B) RNA was extracted by TRI reagent, and semiquantitative RT-PCR was performed using human Elf-3 primer as described in the Materials and Methods section. Each lane shows RNA from an individual mouse and is representative of RT-PCR done on 5 mice in each group. Elf-3/GAPDH RNA was quantitated by scanning densitometry and is represented as percentage change of MMP-9−/− mice compared with WT mice. Values are representative of 3 individual experiments, mean ± SE; n = 5 for WT and MMP-9−/− in each of the experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
MMP-9 expression inversely correlates with goblet cell differentiation. HT29-cl.16E cells were plated on 0.4-mm filters at confluency, and cell lysates and filters were collected on days 1, 3, 5, 7, 10, and 21. (A) Cells were lysed and immunoblotted for MMP-9, NICD, and MUC-2. β-actin was used as the loading control. Representative blots from 2 independent experiments are shown (n = 4). Each lane shows protein (30 μg/lane). (B) One set of filters was fixed and processed for electron microscopy. MG, mucin granules.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
MMP-9 over expression modulates goblet cell-specific gene expression. HT29-cl.16E cells were transfected with pEGFP plasmid with or without the MMP-9 gene, n = 6 per condition as described in the Materials and Methods section. (A) The efficiency of MMP-9 transfection was confirmed by Western blot. Western blot analysis of MUC-2 and the expression of MMP-9, NICD, and KLF-4 in HT29-cl.16E cells were done by collecting supernatants and whole cell lysates, respectively. Equal amounts of protein were separated by SDS-PAGE and probed with the respective antibodies. β-actin served as the loading control. Each lane shows protein (30 μg/lane). Western blot was quantitated by scanning densitometry and represented as percentage change of MUC-2, NICD, and KLF-4/β-actin in MMP-9 over expressed cells compared with vector. Values are representative of 3 individual experiments with 3 samples per group, mean ± SE. (B) HT29-cl.16E cells transfected with vector or MMP-9 were fixed with paraformaldehyde and stained with AB pH 2.5 and PAS. Representative images obtained from 6 samples and 4 fields/sample are shown (original magnification, 20×), and acidic mucin is indicated by arrows.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
Silencing of the MMP-9 gene results in preferential differentiation toward the goblet cell lineage. Caco2-BBE cells were transfected with MMP-9 siRNA, n = 6 per condition as described in the Materials and Methods section. (A) The efficiency of MMP-9 siRNA was confirmed by Western blot. Western blot analyses of MMP-9, NICD, KLF-4, and hPepT1 in Caco2-BBE cells were done by collecting whole cell lysates. Equal amounts of protein were separated by SDS-PAGE and probed with the respective antibodies. β-tubulin served as the loading control. Each lane shows protein (30 μg/lane). Western blot was quantitated by scanning densitometry and represented as percentage change of NICD, hPepT1, and KLF-4/β-tubulin in MMP-9 siRNA transfected cells compared with cells transfected with scrambled RNA. Values are representative of 3 samples per group done in replicates, mean ± SE. (B) RNA was extracted by TRI reagent, and RT-PCR was performed using human MUC-2 and hPepT1 primers as described in the Materials and Methods section. GAPDH served as the loading control. MUC-2 and hPepT1/GAPDH RNA was quantitated by scanning densitometry and is represented as percentage change in MMP-9 siRNA transfected cells compared with cells transfected with scrambled RNA. Values are representative of 3 individual experiments with 3 samples per group, mean ± SE.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
MMP-9 expression modulates S typhimurium adhesion to epithelial cells. HT29-cl.16E cells were seeded on 1-cm2 inserts and were transfected with pEGFP plasmid with or without the MMP-9 gene (A). Similarly seeded HT29-cl.16E cells were transfected with scrambled or MMP-9 siRNA (B) as described in the Materials and Methods section. Cells were subsequently infected with S typhimurium for 1 hour, and the adherence assay was performed as described in the Materials and Methods section. S typhimurium was quantified by plating in serial dilutions for CFU on LB agar medium and incubated overnight at 37°C. Data represent mean ± SE. n = 4, P < .005.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions