1. Volume 127, Issue 2, Pages 607-620 (August 2004)
KLF11 mediates a critical mechanism in TGF-β signaling that is inactivated by Erk- MAPK in pancreatic cancer cells 
Volker Ellenrieder, Anita Buck, Ana Harth, Kerstin Jungert, Malte Buchholz, Guido Adler, Raul Urrutia, Thomas M. Gress 
Gastroenterology 
Volume 127, Issue 2, Pages (August 2004)
DOI: /j.gastro

2. Figure 1
KLF11 enhances TGF-β signaling and transcription. (A) A total of 100 ng of either p3TP-Lux or 3xSBE-Luc was transiently transfected along with 100 ng full-length KLF11 or the parental vector and treated with TGF-β (5 ng/mL) for 24 hours. The luciferase activity was measured and expressed as fold induction by TGF-β. (B) Doxycycline-inducible HEK293 cells were induced to express KLF11, and TGF-β was applied for an additional 60 minutes. Proteins were extracted and immunoblotting was performed using antibodies against KLF11, receptor-phosphorylated Smad2, and total Smad2. (C) KLF11 expression was induced by doxycycline, and its effect on TGF-β-induced collagen-I mRNA expression was determined by Northern blot analysis. Doxycycline-induced expression of KLF11 led to a sustained increase of TGF-β-induced collagen-I transcript levels. The minor variations of RNA loaded to individual lanes as shown by the 28s ribosomal RNA band do not affect the interpretation of the data, which were confirmed by real-time analysis.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
The mSin3a-interacting SID motif is required for KLF11-mediated increase in TGF-β signaling. Cells were transiently transfected with p3TP-Lux along with full-length KLF11wt, KLF11-SIDdel, or KLF11-R2/R3del and treated with TGF-β for 24 hours. Luciferase activity was measured, and mean fold induction values with respect to empty vector control were calculated from 3 independent experiments. Values are mean ± SD. The statistically significant difference between KLF11wt- and KLF11-SIDdel-mediated effects on TGF-β-induced p3TP-Lux reporter activity is indicated by the asterisk (Kruskal-Wallis H test; P < 0.05).
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
KLF11 terminates TGF-β-induced Smad7 expression. HEK293 cells were transfected with full-length KLF11 wild-type. Twenty-four hours after transfection, cells were treated with TGF-β (5 ng/mL) for an additional 3 hours. (A) KLF11-mediated effects on Smad7 mRNA expression were detected by real-time PCR and calculated relative to basal Smad7 mRNA levels per time point. Values were shown as fold expression. (B) KLF11-induced changes of Smad7 protein levels were studied by Western blot analysis using anti-Smad7 antibodies. KLF11 induction was confirmed using anti-KLF11 antibodies, and protein loading was controlled using anti-actin antibodies. (C) KLF11 inhibits TGF-β-induced Smad7 promoter activity. HEK293 cells were transiently transfected with 100 ng pGL725-Luc Smad7 reporter construct and increasing amounts of full-length KLF11 before treatment with TGF-β (5 ng/mL for 24 hours). Luciferase activity was measured and TGF-β-induced reporter activity was expressed as mean fold induction. Mean values were calculated from 3 independent experiments and are shown as mean ± SD. (D) Full-length KLF11wt, KLF11-SIDdel, or KLF11-R2/R3del were transiently cotransfected with the pGL725-Luc Smad7 reporter construct. Luciferase activity was measured and expressed as relative luciferase activity.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
KLF11-mediated repression of the Smad7 promoter requires binding to GC-rich boxes of the proximal Smad7 promoter. (A) Schematic representation of the pGL725 (−603/+112) Smad7 reporter construct including the predicted GC-rich KLF11 binding sites (boxes A-D) and a previously described SBE.26 Cells were transfected with KLF11wt along with either pGL725-Luc, pGL725ΔSBE-Luc, or pGL725ΔGC-Luc Smad7 reporter constructs and incubated in the absence or presence of 5 ng/mL TGF-β for 24 hours. (B) GST-KLF11-ZF and GST alone were used in gel-shift assays with 32P-labeled probes of either wild-type Smad7 promoter boxes A, B, C, or D (wt) or mutant boxes in which the individual KLF11 binding sites are inactivated (mut). The specific complex that forms between GST-KLF11-ZF and GC-rich boxes is indicated by the arrow. (C) The proximal Smad7 promoter (region −129 to −209 base pairs) was cloned into the pGL3-basic luciferase vector (pGL80) and the GC boxes were individually or combined mutated (pGL80M[a], pGL80M[b], pGL80M[c/d], or pGL80M[a-d]). The functional relevance of the individual GC boxes in KLF11-mediated repression was verified in transient reporter assays.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
(A) Smad7 and KLF11 mRNA expression in tissues from pancreatic ductal adenocarcinomas (n = 16) and controls (n = 6). As control pancreas, we used 2 pancreatic tissue samples (numbers 5 and 6) from organ donors and 4 tumor-free resection margins from patients with pancreatic cancer (numbers 1–4). RNA was extracted and Smad7 and KLF11 mRNA levels were measured by real-time PCR and calculated relative to the mean values measured for control pancreas. (B) KLF11-mediated Smad7 repression is inhibited in pancreatic cancer cells. A total of 100 ng of the pGL725-Luc Smad7 reporter plasmid was transfected into PANC-1, MiaPaca2, and Colo357 cells along with either 100 ng of the parental vector or full-length KLF11. The luciferase activity was measured and expressed as relative luciferase activity. The lower panel shows similar levels of KLF11 protein expression after successful transfection. (C) PANC-1 cells were transfected with pGL725 and increasing amounts of KLF11. On transfection, PANC-1 cells were pretreated with PD98059 before application of TGF-β. Note that KLF11 repression activity was restored by inhibition of Erk activation.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
(A) Erk-MAPK induces KLF11 phosphorylation and disrupts binding to the mSin3A corepressor in pancreatic cancer cells. PANC-1 cells were transfected with either the parental vector or FLAG-tagged KLF11, followed by treatment with PD98059 and metabolic labeling with [32P]orthophosphate. Immunoprecipitation was performed with anti-FLAG antibodies, and phosphorylation of KLF11 was detected by autoradiography. KLF11 expression and Erk activation were monitored by Western blotting using specific antibodies. (B) Total Erk2 from either PD98059-treated or untreated PANC-1 cell extracts was immunopurified using anti-Erk2 antibodies and incubated with GST-KLF11 to phosphorylate GST-KLF11 in vitro. Immunoprecipitated Erk was subjected to Western blot analysis using anti-phospho-Erk and anti-total Erk antibodies and immunoblotting using anti-KLF11 antibodies to control for KLF11 expression. (C) KLF11 was immunoprecipitated from transfected PANC-1 cells using anti-FLAG antibodies, and binding of endogenous mSin3a was detected by immunoblotting using anti-Sin3a antibodies. Note that KLF11 weakly binds endogenous mSin3a (lane 2) in untreated PANC-1 cells, whereas pretreatment with PD98059 restores mSin3a binding (lane 3).
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Mutation of Erk consensus site restores KLF11-mediated repression of Smad7 and enhances activity of the Smad pathway in pancreatic cancer cells. (A) A schematic representation of KLF11 protein structure including the 4 MAPK phosphorylation sites (PLT56P, PQS94P, PVS107P, and PSS149P), which were jointly mutated to alanine in the context of the Flag-tagged KLF11 full-length construct (esmc). (B) PANC-1 cells and MiaPaca2 cells were transfected with pGL725 together with either KLF11wt or KLF11-ESMC, and repression of the Smad7 pGL725 promoter was verified in transient reporter assays. (C) The effects of KLF11wt and KLF11-ESMC on Smad7 mRNA expression were determined by real-time PCR, calculated relative to basal Smad7 mRNA levels and expressed as fold expression. (D) PANC-1 cells were transfected with either KLF11wt or KLF11-ESMC, and their effects on endogenous Smad7 expression and mSin3A binding were determined using cell lysates and immunoprecipitates, respectively. Note that the Erk-insensitive KLF11-ESMC mutant efficiently interacts with endogenous mSin3a (top panel) and inhibits endogenous Smad7 expression in PANC-1 cells. Controls show a detectable change in the apparent mobility of unphosphorylated KLF11-ESMC as compared with KLF11wt.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
Prevention of Erk-induced phosphorylation restores KLF11-mediated enhancement of Smad signaling in pancreatic cancer cells. A total of 100 ng of either (A) 3xSBE-Luc or (B) p3TP-Lux was transfected into PANC-1 cells along with KLF11wt or KLF11-ESMC and treated with TGF-β for 24 hours. Luciferase activity was measured and mean fold-induction values with respect to empty vector control were calculated from 3 independent experiments. Values are shown as mean ± SD. Note that mutation of Erk phosphorylation sites rescued KLF11-induced increase of Smad signaling and transcription.
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 9
Flow chart describing the mechanism of the KLF11-mediated increase of Smad signaling activity in normal epithelial cells and its inhibition by oncogenic Ras-Erk signaling in pancreatic cancer cells. Induction of KLF11 by TGF-β causes silencing of the Smad7 gene and thus terminates the autoinhibitory feedback loop imposed by Smad7. Activation of the oncogenic Ras-Mek-Erk MAPK pathway leads to phosphorylation-induced inactivation of KLF11 and therefore antagonizes the ability of KLF11 to enhance Smad signaling activity. These findings suggest a novel link for a crosstalk between oncogenic Ras and the TGF-β pathway that is defined by Erk-induced inactivation of the Smad signaling enhancer protein KLF11. Arrows denote up-regulation or activation, and T bars denote down-regulation or inactivation.
Gastroenterology  , DOI: ( /j.gastro )