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First published online 7 October 2008
doi: 10.1242/jcs.032706

Journal of Cell Science 121, 3531-3540 (2008)
Published by The Company of Biologists 2008
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Lkb1 is required for TGFβ-mediated myofibroblast differentiation

Kari Vaahtomeri1, Eeva Ventelä1,*, Kaisa Laajanen1,*, Pekka Katajisto1,*, Pierre-Jean Wipff2, Boris Hinz2, Tea Vallenius1, Marianne Tiainen1,{ddagger} and Tomi P. Mäkelä1,§

1 Genome-Scale Biology Program, Institute of Biomedicine, Biomedicum Helsinki, P.O. Box 63, 00014 University of Helsinki, Finland
2 Laboratory of Cell Biophysics, Bâtiment SG-AA-B143, Station 15, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland


Figure 1
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  		Fig. 1. Lkb1 modulates TGFβ signaling. (A) Fold induction of luciferase activity in MEFs with the indicated Lkb1 genotypes following infection with AdCre or AdLacZ (control) recombinant adenoviruses and subsequently transfected with either (CAGA)12-luciferase, ARE-luciferase or BRE-luciferase plasmids and RL-TK to correct for transfection efficiency. Fold induction is indicated relative to control samples and all bars represent averages of three experiments performed with independently isolated MEFs using triplicate samples, except for the (CAGA)12-luc Lkb1lox/+ and (CAGA)12-luc Lkb1lox/lox bars, which represent averages from ten and five experiments, respectively. Right panel shows similar analysis with MEFs transfected with non-targeting (control) or Lkb1-targeting (siLkb1) siRNAs and (CAGA)12-luc. Bars represent averages from two experiments using four to ten samples per experiment. Error bars indicate s.d.; **P<0.01, ***P<0.001. (B) Western blotting analysis of Lkb1, activated Smad2 (Smad2-P, shown as p-Smad2) and total Smad2 from serum-starved (0.2% FCS) untreated (–) or TGFβ-treated (+; 1 ng/ml) control (AdLacZ-infected Lkb1lox/– MEFs) or Lkb1–/– (AdCre infected Lkb1lox/– MEFs) MEFs. A background band in the Smad2-P blot is indicated with an asterisk. (C) Immunofluorescence staining using an antibody recognizing Smad2 and Smad3 of serum-starved (0% FCS, 22 hours) and subsequently untreated (–) or TGFβ-treated (1 ng/ml) control and Lkb1–/– MEFs. The apparently lower total intensity of signal might be due to more diffuse localization of Smad2 and Smad3 in the cytoplasm of Lkb1–/– MEFs compared with controls because no difference in total levels were noted in western blotting (see Fig. 1B). Nuclei are visualized with Hoechst. A single-cell magnification is shown in the inset. (D) (CAGA)12-luc activity in control and Lkb1–/– MEFs transfected with constitutively active ALK5 (ca-ALK5) or vector control and treated with added TGFβ as indicated. The value of relative (CAGA)12-luc activity represents the ratio of (CAGA)12-luc to Renilla-luciferase–thymidine-kinase (RL-TK), with the average from a representative experiment out of three using triplicate samples shown; error bars indicate s.d.

 

Figure 2
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  		Fig. 2. Myofibroblast marker expression is dependent on Lkb1. (A) SMA immunofluorescence staining of MEFs treated for 72 hours with TGFβRI inhibitor (SB431542), indicating a requirement for TGFβ signaling in SMA expression by primary MEFs. (B,C) Staining of SMA (B) and SM22 (C) in control and Lkb1–/– MEFs, demonstrating decreased myofibroblast marker expression in the absence of Lkb1. Nuclear Hoechst stains are shown to demonstrate comparable cell numbers. (D) Phalloidin and vinculin staining, demonstrating a decrease in the amount of stress fibers and the mislocalization of focal adhesions in Lkb1–/– MEFs. (E) Quantitation of the percentage of wrinkling cells in each genotype, showing the average out of three experiments. Error bars indicate s.d.; *P<0.05.

 

Figure 3
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  		Fig. 3. Unaltered FAK and SRF activity in Lkb1–/– MEFs. (A) FAK activity measured by anti-phosphotyrosine (p-Tyr) immunoblot signals from anti-FAK immunoprecipitates (Owen et al., 1999Go) after 50 minutes of adhesion on adhesive fibronectin (FN) plates or non-adhesive BSA-coated plates. Below are total cell extracts used for immunoprecipitations to indicate similar FAK levels between control (indicated by `C') and Lkb–/– immunoprecipitates. (B) SRE-luc activity in semiconfluent control and Lkb1–/– MEFs following a 24-hour serum starvation (0% FCS) or subsequent 3- or 15-hour treatment with serum (20% FCS) or TGFβ. Corrected luciferase values are normalized to the 0% FCS sample of control MEFs. The averages from five experiments using triplicate samples are shown, except for the 15-hour treatments, which represent averages from three experiments. Error bars indicate s.d.; *P<0.05. (C) Western blotting analysis of control and Lkb1–/– MEFs was performed with anti-moesin-P (p-Moesin), anti-PKB-P (p-PKB) and anti-SMA antibodies.

 

Figure 4
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  		Fig. 4. Exogenous TGFβ rescues the defective myofibroblast differentiation of Lkb1–/– MEFs. (A) Immunofluorescence staining for SMA of semiconfluent control and Lkb1–/– MEFs following serum starvation (0.2% FCS), as well as subsequent treatment with TGFβ, demonstrating a rescue of SMA staining upon TGFβ treatment. (B) Lysates from non-treated and TGFβ-stimulated control (indicated by `C') and Lkb1–/– MEFs were analyzed by western blotting with antibodies specific for SMA, SM22 or CDK7 (a loading control). Lanes have been reorganized, as indicated by the black bar. (C) Immunofluorescence staining for vinculin (green) and phalloidin (red) of control and Lkb1–/– MEFs, demonstrating recovery of mature focal adhesions and stress fibers in TGFβ-treated Lkb1–/– MEFs. (D) SMA-luc activity following 20 hours of TGFβRI inhibitor (SB431542) treatment, 48 hours after Smad7 transfection or upon deletion of one (Lkb1+/–) or both (Lkb1–/–) alleles of Lkb1. DMSO-treated (left), vector-transfected or control-infected samples are shown with white bars. Fold induction is indicated relative to these controls. Bars represent the average out of three experiments using triplicate samples, except for the Lkb1+/– and Lkb1–/– bars, which represent averages of nine and four experiments, respectively. Error bars indicate s.d.; *P<0.05, **P<0.01, ***P<0.001. (E) SMA-luc and SM22-luc activity in control or Lkb1–/– (AdCre) MEFs with added TGFβ as indicated. Fold induction of corrected luciferase values of non-treated and TGFβ-treated Lkb1–/– MEFs to non-treated control samples are shown. Bars represent averages from three experiments using triplicate samples. Error bars indicate s.d.; *P<0.05, ***P<0.001.

 

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© The Company of Biologists Ltd 2008