Krüppel-like Factor 5 Plays an Important Role in the Pathogenesis of Chronic Pancreatitis

Abstract

Chronic pancreatitis results in the formation of pancreatic intraepithelial neoplasia (PanIN) and poses a risk of developing pancreatic cancer. Our previous study demonstrated that Krüppel-like factor 5 (KLF5) is necessary for forming acinar-to-ductal metaplasia (ADM) in acute pancreatitis. Here, we investigated the role of KLF5 in response to chronic injury in the pancreas. Human tissues originating from chronic pancreatitis patients showed increased levels of epithelial KLF5. An inducible genetic model combining the deletion of Klf5 and the activation of Kras^G12D mutant expression in pancreatic acinar cells together with chemically induced chronic pancreatitis was used. The chronic injury resulted in increased levels of KLF5 in both control and Kras^G12D mutant mice. Furthermore, it led to numerous ADM and PanIN lesions and extensive fibrosis in the KRAS mutant mice. In contrast, pancreata with Klf5 loss (with or without Kras^G12D) failed to develop ADM, PanIN, or significant fibrosis. Furthermore, the deletion of Klf5 reduced the expression level of cytokines and fibrotic components such as Il1b, Il6, Tnf, Tgfb1, Timp1, and Mmp9. Notably, using ChIP-PCR, we showed that KLF5 binds directly to the promoters of Il1b, Il6, and Tgfb1 genes. In summary, the inactivation of Klf5 inhibits ADM and PanIN formation and the development of pancreatic fibrosis.

Keywords: Kirsten rat sarcoma viral oncogene homolog; Krüppel-like factor 5; acinar-to-ductal metaplasia; pancreatic intraepithelial neoplasia.

Figure 1

Representative immunohistochemistry images of KLF5 expression in the pancreas of chronic pancreatitis patients (magnification 10×).

Figure 2

Deletion of Klf5 in pancreatic acinar cells results in the inhibition of chronic pancreatitis in a murine model of this disease. Representative images of H&E staining of the pancreas of normal and chronic pancreatitis mice. Ptf1aCre^ERTM;Rosa26^tdTomato/+, Ptf1aCre^ERTM;Rosa26^tdTomato/+;Klf5^fl/fl, Ptf1aCre^ERTM;Rosa26^tdTomato/+;Kras^G12D, and Ptf1aCre^ERTM;Rosa26^tdTomato/+;Kras^G12D;Klf5^fl/fl mice were treated with cerulein for one, two, three, and four weeks or with PBS for four weeks. CER—cerulein. Scale bar = 200 µm.

Figure 3

Deletion of Klf5 in pancreatic acinar cells reduces injury during chronic pancreatitis in a murine model of this disease. Ptf1aCre^ERTM;Rosa26^tdTomato/+, Ptf1aCre^ERTM;Rosa26^tdTomato/+;Klf5^fl/fl, Ptf1aCre^ERTM;Rosa26^tdTomato/+;Kras^G12D, and Ptf1aCre^ERTM;Rosa26^tdTomato/+;Kras^G12D;Klf5^fl/fl mice were treated with cerulein or PBS for four weeks. CER—cerulein. (A) Quantification of pancreatic damage score per pancreas after cerulein treatment (n = 6). (B) ADM scores, (C) mPanIN score, (D) edema, (E) inflammatory infiltration, (F) loss of pancreatic ducts per pancreas from female mice after cerulein treatment. * p < 0.05, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA test (Data represent mean ± S.D.). Quantification of serum amylase (G) and lipase (H) from cerulein-treated and PBS-treated mice (n = 3). *** p < 0.001 by t-test (Data represent mean ± S.D.).

Figure 4

KLF5 levels are increased in the murine model of chronic pancreatitis. Immunohistochemical analysis of KLF5 expression in pancreatic tissues from female mice of indicated genotypes and treatments. Scale bars = 200 µm. Arrows show examples of KLF5-positive staining.

Figure 5

Genetic inactivation of Klf5 inhibits the transformation of acinar cells to ADM following chronic injury. Multicolor immunofluorescence (IF) analysis of amylase (green), Keratin-19 (red), and nuclei (Blue) in pancreatic tissues from mice of indicated genotypes and treatments. Scale bar = 200 µm.

Figure 6

Genetic inactivation of Klf5 inhibits PanIN formation following chronic injury. Alcian blue staining counterstained with Nuclear Fast Red of pancreatic tissue from mice of indicated genotypes and treatments. Scale bar = 200 µm. * marks positive Alcian blue stain in Ptf1aCre^ERTM;Rosa26^tdTomato/+;Klf5^fl/fl mice.

Figure 7

Genetic inactivation of Klf5 in vivo suppresses pancreatic stellate cell activation. Representative images of multicolor immunofluorescence (IF) analysis of alpha-SMA (green), Keratin-19 (red), and nuclei (Blue) in pancreatic tissues from mice of indicated genotypes and treatments. Scale bar = 200 µm.

Figure 8

Genetic inactivation of Klf5 in vivo suppresses fibrosis. Representative images of Masson’s trichrome stain of mice of indicated genotypes and treatment. Scale bar = 200 µm.

Figure 9

KLF5 regulates the expression of inflammatory and fibrotic markers during chronic pancreatitis. (A) qRT-PCR results of gene expression of Il6, Il1b, Tgfb1, Tnf, Mmp9, Timp1, Col1a1, and Acta2 in the whole pancreatic tissues from mice of indicated genotypes and treatments. ** p < 0.01, *** p < 0.001, **** p < 0.0001, from one-way ANOVA test (Data represent mean ± S.D.). (B) qRT-PCR results of gene expression of Il6, Il1b, Tgfb1, and Tnf in tdTomato-positive cells originated from pancreatic tissues from mice of indicated genotypes and treatments. * p < 0.05, ** p < 0.01 from one-way ANOVA test (n = 3, data represent mean ± S.D.). (C) Relative activity of Il1b, Il6, and Tgfb1 promoters. HEK 293T cells were co-transfected with control plasmid or plasmid encoding KLF5 and either of the promoters. Twenty-four hours after transfection, the activity of the promoters was measured. ** p < 0.01, *** p < 0.001, **** p < 0.0001 by t-test (n = 6, Data represent mean ± SD). (D) Predicted binding sites (yellow) for KLF5 in sequence 1.5 kb upstream of the translation start site of the Il1b, Il6, and Tgfb1 promoters using the JASPAR database. T.S.S.—transcription start site. (E) PCR amplification of site-specific sequences of DNA. Product of ChIP from Il1b, Il6, and Tgfb1 promoters using an anti-KLF5 antibody.

Figure 10

KLF5 binds to the promoters of Il6, Il1b, and Tgfb1 during chronic pancreatitis. PCR amplification of site-specific sequences of DNA. Product of ChIP-PCR from (A–D) Il1b, Il6, and Tgfb1 promoters using an anti-KLF5 antibody.

Figure 11

Deletion of Klf5 in vivo inflammatory response upon chronic pancreatitis. (A) Mouse cytokine arrays showing the levels of proteins from pancreatic tissues of Ptf1aCre^ERTM; Rosa26^tdTomato/+ and Ptf1aCre^ERTM; Rosa26^tdTomato/+; Klf5^fl/fl mice treated with cerulein for four weeks. The boxes mark differentially expressed proteins. (B) Quantification of the levels of differentially expressed proteins from (A).