Keratinocyte growth factor promotes goblet cell differentiation through regulation of goblet cell silencer inhibitor  Dai Iwakiri, Daniel K. Podolsky  Gastroenterology  Volume 120, Issue 6, Pages 1372-1380 (May 2001) DOI: 10.1053/gast.2001.24029 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Ligand stimulated tyrosine phosphorylation of KGFR in H2-UD cells. Cell lysates from either untreated cells or cells treated with 50 ng/mL KGF for 10 minutes were subjected to Western blot analysis with anti-KGFR (C-17) antibody (left panel) or antiphosphotyrosine (right panel) antibody. Molecular weight markers are indicated. Gastroenterology 2001 120, 1372-1380DOI: (10.1053/gast.2001.24029) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 KGF induces the expression of ITF and MUC 2 mRNA in H2-UD cells. H2-UD cells were treated with 50 ng/mL KGF for various times indicated at the top of panels and total RNA was extracted. Human (A) ITF mRNA, (B) MUC2 mRNA, and (C) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were detected by sequential probes in Northern blot analysis as detailed in Materials and Methods. Basal mRNA expression of ITF and MUC2 of H2-D cells are shown in the right panel. Gastroenterology 2001 120, 1372-1380DOI: (10.1053/gast.2001.24029) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 KGF induces ITF protein synthesis in H2-UD cells. (A) Western blot analysis of ITF protein synthesis in H2-UD cells untreated or treated with 50 ng/mL KGF for various times as indicated above the panel. Cell lysates from each untreated or treated cell were subjected to Western blot, and ITF protein (arrow) was detected by anti-ITF antibody. (B) Concentration-dependent induction of ITF protein synthesis by KGF. H2-UD cells were treated with KGF (0, 1, 10, and 100 ng/mL) and cell lysates were developed by Western blot analysis. ITF protein indicated by the arrow. Gastroenterology 2001 120, 1372-1380DOI: (10.1053/gast.2001.24029) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Effects of KGF on ITF gene promoter transcription. (A) Model of mITF cis-acting elements. The regulatory elements including enhancer and silencer elements identified in previous studies and their locations in mITF 5'-flanking region are indicated within the box. E and S indicate enhancer and silencer elements, respectively. (B) Reporter plasmid constructs were used for transient transfection assays. The mITF/luciferase (LUC) reporter plasmid construct 6353WT and shorter deletion construct 1848WT generated from 6353WT were composed of 5' flanking sequence from murine ITF gene ligated to the luciferase promoter. The nucleotide positions of mITF gene are also indicated. (C) Transient transfection assays of reporter plasmid constructs in H2-UD cells untreated or treated with 10 ng/mL KGF. ‘0’ indicates no KGF treatment. The normalized transcriptional activity (luciferase/β-galactosidase units) (see Materials and Methods) from 6353WT (▨) or 1848WT (■) construct in each transfected cell is shown. All transfections were done in triplicate and repeated at least 3 times. Results are shown as the mean ± SE. Gastroenterology 2001 120, 1372-1380DOI: (10.1053/gast.2001.24029) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 KGF regulation of ITF promoter activity through the GCSI element. (A) Shorter reporter plasmid constructs of mITF gene. The shorter internal deletion constructs of the mITF gene were generated as described in Materials and Methods. The construct designated as WT has one silencer (indicated by S) and enhancer (indicated by E), GCRE, and GCSI element described in Figure 4A. The nucleotide positions of mITF gene are indicated. GCSI mut construct also contains one silencer and enhancer, GCRE, and mutant GCSI element. Both wild-type ( GCSI WT) and mutated GCSI ( GCSI mut) sequences are described above. The X shown in this construct scheme indicates mutation of GCSI. (B) Transient transfection assays of WT and GCSI mut construct in H2-UD cells untreated (0 ng/mL) or treated with 10 ng/mL KGF. Normalized transcriptional activity (luciferase/β-galactosidase units) from WT (■) or GCSI mut (□) construct in each transfected cell is shown. All transfections were done in triplicate and repeated at least 3 times. Results are shown in the mean ± SE indicated by error bars. Gastroenterology 2001 120, 1372-1380DOI: (10.1053/gast.2001.24029) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 EMSA of GCSI binding proteins in goblet-like phenotype cell lines and H2-UD cells. (A) EMSAs were carried out with radiolabeled oligonucleotide probe corresponding to the sequence of GCSI. Nuclear proteins were extracted from LS174T, H2-D, and H2-UD cells nontreated or treated with 50 ng/mL KGF for 24 hours. Cold oligonucleotides were also used as the competitor. GCSI-BP and free DNA probes are indicated by arrows. (B) EMSA of GCSI-BP in nuclear extracts prepared from KGF-treated H2-UD cells. Cells were treated with 50 ng/mL KGF for various times as described and EMSA carried out. GCSI-BP and free DNA probes are shown by arrows. Gastroenterology 2001 120, 1372-1380DOI: (10.1053/gast.2001.24029) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Down-regulation of KGFR expression associated with goblet cell differentiation. Western analysis of KGFR expression in H2-D and H2-UD cells. Cell lysates from both cells were subjected to immunoblotting using anti-KGFR antibody (C-17). KGFR protein of each cell indicated by bar. (B) Cell lysates from H2-UD cells treated with 50 ng/mL KGF for various times (0, 24, 48, and 72 hours) were subjected to Western analysis. Bars indicate KGFR proteins and molecular weight markers. Gastroenterology 2001 120, 1372-1380DOI: (10.1053/gast.2001.24029) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of KGF on ITF transcription in H2-D cells. Transient transfection assays of reporter plasmid construct 6353WT in H2-D cells untreated or treated with 10 ng/mL KGF. ‘0’ indicates no KGF treatment. Normalized transcriptional activity (luciferase/β-galactosidase units) is shown. All transfections were done in triplicate and repeated at least 3 times. Results are shown as the mean ± SE indicated by error bars. Gastroenterology 2001 120, 1372-1380DOI: (10.1053/gast.2001.24029) Copyright © 2001 American Gastroenterological Association Terms and Conditions