1. Volume 139, Issue 6, Pages 2124-2134.e9 (December 2010)
Klf4 Overexpression Activates Epithelial Cytokines and Inflammation-Mediated Esophageal Squamous Cell Cancer in Mice 
Marie–Pier Tetreault, Mei–Lun Wang, Yizeng Yang, Jenna Travis, Qian–Chun Yu, Andres J. Klein–Szanto, Jonathan P. Katz 
Gastroenterology 
Volume 139, Issue 6, Pages e9 (December 2010)
DOI: /j.gastro
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2. Figure 1
ED-L2/Klf4 mice develop hyperplasia, inflammation, dysplasia, and cancer. Compared with the (A) 3-month-old littermate controls, (B) H&E-stained esophageal epithelia appeared grossly normal in 3-month-old ED-L2/Klf4 mice. (C and D) At 6 months of age, ED-L2/Klf4 mice showed abnormalities of esophageal epithelial homeostasis. (C) Although basal cells in control mice were homogenous, with densely packed basophilic nuclei, (D) the basal layers of ED-L2/Klf4 mice were expanded and irregular, with nuclear atypia. Marked inflammatory infiltrates also were observed. (E) At 2 years of age, compared with controls, which had no histologic abnormalities, (F) 3 of 6 ED-L2/Klf4 mice developed invasive esophageal squamous cell cancer. Scale bars, 50 μm.
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3. Figure 2
ED-L2/Klf4 esophageal epithelial cells show changes in proliferation but appear to differentiate normally. (A and B) At 6 months of age, KLF4 (red) was not expressed in proliferating cells labeled with BrdU (green) in (A) control and (B) ED-L2/Klf4 mice. (C and D) As compared with (C) controls, the proliferative zone, delineated by staining for keratin 14, which marks proliferating keratinocytes, was greatly expanded in (D) ED-L2/Klf4 mice. (E and F) Keratin 4 staining indicated that keratinocytes entered the differentiation program in both (E) control and (F) ED-L2/Klf4 mice. Scale bars, 10 μm.
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4. Figure 3
KLF4 is absent from esophageal squamous cell cancers in ED-L2/Klf4 mice. (A–C) KLF4 was found predominantly in suprabasal cells in (A) 2-year-old control mice. No KLF4 staining was seen within tumors from (B) ED-L2/Klf4 mice, despite positive staining in (C) esophageal epithelia adjacent to the tumors. (D and E) Staining with Ki-67 revealed proliferation confined to the basal layer of (D) 2-year-old control mice, whereas staining was seen throughout the tumors in (E) ED-L2/Klf4 mice, indicating that these tumors were highly proliferative. Scale bars, 50 μm.
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5. Figure 4
ED-L2/Klf4 mice have infiltration of immune cells into the esophageal mucosa. (A and B) Staining with antibody against the 7/4 antigen revealed no neutrophils in esophageal mucosa of (A) control mice, but a large neutrophilic infiltrate was seen in the esophageal epithelia and lamina propria of (B) ED-L2/Klf4 mice. (C and D) F4/80 staining for macrophages showed no macrophages in (C) control esophageal mucosa, whereas (D) ED-L2/Klf4 mice had macrophages mostly within basal epithelial and subepithelial cells. (E and F) CD3, a marker of T cells, was absent from (E) control esophageal mucosa, but T cells were observed infiltrating the lamina propria and epithelia of (F) ED-L2/Klf4 mice. Scale bars, 50 μm.
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6. Figure 5
Klf4 overexpression leads to cytokine induction in esophageal keratinocytes. (A) By quantitative real-time PCR, CXCL5 mRNA expression was increased in esophagus of ED-L2/Klf4 mice, compared with controls, by 2.9-fold at 3 months of age and by 100-fold at 6 months of age. (B) TNFα mRNA was similarly induced in esophageal epithelia of ED-L2/Klf4 mice by 3.6-fold and 13-fold at 3 and 6 months of age, respectively. (C) In primary esophageal keratinocytes from ED-L2/Klf4 mice, CXCL5 and TNFα mRNA expression were increased by 12- and 31-fold, respectively, compared with primary keratinocytes from control mice. (D) Induction of G-CSF, by 9-fold, and IL-1α, by 17-fold, in primary esophageal keratinocytes from ED-L2/Klf4 mice was confirmed at the mRNA level using real-time quantitative PCR. (E) Moreover, a 44-fold increase in mRNA expression of G-CSF was seen in the esophagus of 6-month-old ED-L2/Klf4 mice. (F) IL-1α mRNA was up-regulated 2–fold in ED-L2/Klf4 mouse esophagus. No changes in expression of G-CSF or IL-1α mRNA were found in the esophagus at 3 months of age. *P < .02.
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7. Figure 6
NF-κB activation in esophageal keratinocytes mediates cytokine induction by KLF4. (A) NF-κB transcriptional activity, assessed with an NF-κB luciferase reporter, was increased in primary esophageal keratinocytes from ED-L2/Klf4 mice compared with control keratinocytes. This increase was blocked by treatment with IKK2 inhibitor. (B–E) As determined by quantitative real-time PCR, mRNA induction of (B) CXCL5, (C) TNFα, (D) G-CSF, and (E) IL-1α in ED-L2/Klf4 keratinocytes was blocked by treatment with IKK2 inhibitor. *Significant difference from control (+dimethyl sulfoxide [dmso]) at a P value of less than .05. **Significant difference from ED-L2/Klf4 (+DMSO) at a P value of less than .05. (F) When primary esophageal keratinocytes from control and ED-L2/Klf4 mice were treated with IKK2 inhibitor, cell viability, as assessed by Trypan blue exclusion, was decreased significantly only in ED-L2/Klf4 keratinocytes with inhibition of NF-κB signaling. *Significant difference from control at a P value of less than .05 (n = 4).
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8. Figure 7
TNFα is not required for induction of CXCL5, G-CSF, and IL-1α in primary esophageal keratinocytes. (A) Treatment of primary esophageal keratinocytes from ED-L2/Klf4 mice with TNFα neutralizing antibody inhibited NF-κB transcriptional activity. (B–E) Quantitative real-time PCR revealed that treatment of ED-L2/Klf4 keratinocytes with TNFα neutralizing antibody failed to block the induction of (B) CXCL5, (C) TNFα, (D) G-CSF, and (E) IL-1α mRNA. *Significantly different from control (no treatment) at a P value of less than .05. **Significantly different from ED-L2/Klf4 (no treatment) at a P value of less than .05. (F) A model for the role of KLF4 in inflammation, the epithelial barrier, and squamous cell carcinogenesis of the esophagus.
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9. Supplementary Figure 1
Transgenic expression of Klf4 in the esophagus of ED-L2/Klf4 mice. (A) Ribonuclease protection assays showed strong transgene expression in the esophagus (Eso), with weak expression in the stomach (Sto), likely the squamous forestomach, and no expression in the duodenum (Duod) or liver (Liv). As expected, endogenous Klf4 was seen in the esophagus, stomach, and duodenum, but not the liver. Cyclophilin was used as a loading control. (B and C) Although nuclear KLF4 staining was seen throughout the suprabasal and superficial layers in (B) 1-month-old control mice, KLF4 staining in (C) ED-L2/Klf4 mice appeared stronger, and nuclear staining was seen in more cells, including in cells of the basal layer. Scale bars, 50 μm. (D) At 3 months of age, compared with controls (Cont), ED-L2/Klf4 mice (Mut) had increased KLF4 by Western blot. However, at 6 months of age, KLF4 was decreased slightly in ED-L2/Klf4 mice compared with controls, likely the result of the observed expansion of the basal layer, where KLF4 was not expressed. (E) By Western blot, KLF5 was decreased in ED-L2/Klf4 mice at 3 months of age but increased at 6 months of age. This mirrored the findings with KLF4. (F and G) Compared with (F) littermate controls, (G) 6-month-old ED-L2/Klf4 mice had increased nuclear KLF5 staining in esophageal epithelia. Scale bars, 50 μm.
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10. Supplementary Figure 2
Lower-power views of (A and C) control and (B and D) ED-L2/Klf4 mice at (A and B) 3 months and (C and D) 6 months of age revealed hyperplasia, inflammation, and dysplasia in 6-month-old ED-L2/Klf4 mice. Scale bars, 50 μm.
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11. Supplementary Figure 3
Compared with (A and C) littermate controls, (B and D) 6-month-old ED-L2/Klf4 mice had inflammation and hyperplasia in their (A and B) tongues and (C and D) forestomachs. Scale bars: (A and B) 50 μm, (C and D) 10 μm.
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12. Supplementary Figure 4
Quantitation of BrdU-labeled cells revealed a 3.8-fold increase of the number of proliferating cells in the basal layer of 6-month-old ED-L2/Klf4 mice, compared with controls (*P < .002). Proliferation appeared to be slightly increased in 3-month-old mice but did not achieve statistical significance (P = .08).
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13. Supplementary Figure 5
ED-L2/Klf4 mice have dilated intercellular spaces (spongiosis) in esophageal epithelia. (A and B) Compared with (A) controls, esophageal epithelia of (B) 3-month-old ED-L2/Klf4 mice showed intercellular edema in the suprabasal layers, as assessed by transmission electron microscopy. These changes were seen in all ED-L2/Klf4 mice examined (n = 3) and none of the control mice (n = 3). Scale bars, 2 μm. (C) By quantitative real-time PCR, claudin-14, claudin-15, occludin, and desmocollin-2 mRNA expression levels were increased in esophagus of ED-L2/Klf4 mice, compared with controls, by 3.2-fold, 2.7-fold, 1.6-fold, and 2.3-fold, respectively, at 3 months of age.
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14. Supplementary Figure 6
Esophageal mucosa of (A) control mice contained no B cells, as indicated by staining for CD45R, but B cells were present in the esophageal epithelia and lamina propria of (B) ED-L2/Klf4 mice. Scale bars, 50 μm.
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15. Supplementary Figure 7
Wild-type mouse primary esophageal keratinocytes infected with Klf4-expressing retrovirus had increased TNFα (3.8-fold), CXCL5 (2.7-fold), G-CSF (2.4-fold), and IL-1α (2.5-fold) mRNA, compared with control, by quantitative real-time PCR (*P < .01).
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16. Supplementary Figure 8
By mouse cytokine antibody arrays, Klf4 overexpression in primary esophageal keratinocytes increased the release of different proinflammatory cytokines, including TNFα, G-CSF, and IL-1α (red circles). The template of the array is shown at the top. Pos, positive control; Neg, negative control. Data are representative of 2 separate sets of experiments. GM-CSF, Granulocyte-macrophage colony-stimulating factor; M-CSF, Macrophage colony-stimulating factor; MIG, Monokine induced by gamma interferon; MIP-1α, Macrophage inflammatory protein-1α.
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17. Supplementary Figure 9
By MTT assay, naive, wild-type mouse keratinocytes treated with conditioned media from ED-L2/Klf4 keratinocytes had increased proliferation compared with cells treated with media from keratinocytes of control mice.
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18. Supplementary Figure 10
(A) In esophageal epithelia from 3-month-old control mice, nuclear staining for the p65 subunit of NF-κB, indicative of NF-κB activation, was minimal. (B) In contrast, ED-L2/Klf4 mice had extensive nuclear p65 in epithelial cells in the basal and suprabasal layers. (C) By Western blot, phospho-p65, but not total p65, was increased in esophageal epithelia of 3-month-old ED-L2/Klf4 mice compared with controls. (D and E) At 6 months of age, compared with (D) littermate controls, nuclear p65 staining was much more intense in (E) ED-L2/Klf4 mice and was observed both in epithelial and inflammatory cells. Scale bars, 50 μm.
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19. Supplementary Figure 11
(A) NF-κB luciferase reporter assays revealed a 1.8-fold increase (*P = .037) when wild-type mouse primary esophageal keratinocytes were infected with Klf4-expressing retrovirus, compared with control. (B) TNFα neutralizing antibody inhibited NF-κB transcriptional activity in wild-type mouse primary esophageal keratinocytes (*P < .01). Cells were treated with mouse recombinant TNFα in the presence or absence of TNFα neutralizing antibody.
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