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. Author manuscript; available in PMC: 2018 May 11.
Published in final edited form as: Cell Rep. 2018 Apr 10;23(2):442–458. doi: 10.1016/j.celrep.2018.03.067

Figure 7. FOXF1 Regulates Myofibroblast Invasion through Transcriptional Activation of CDH2 and Repression of CDH11.

Figure 7

(A) Schematic drawing of the mouse Cdh2 promoter region with potential FOXF1 DNA binding sites (black boxes). See also Table S2. ChIP assays in NIH 3T3 cells showed direct binding of FOXF1 to the indicated regions in Cdh2 promoter. ChIP assay of FOXF1 binding to the −1,435/−1,428 bp mouse Pdgfb promoter region was used as a positive control. Gapdh DNA, which lacks potential FOXF1 binding motifs, was used as a negative control. FOXF1 binding is shown relative to immunoglobulin G (IgG). Data represent one of two independent experiments.

(B) Schematic drawing of the pGL2-mCdh2-Luc construct containing the Cdh2 promoter region. The mouse Cdh2 promoter (−3,087/+71 bp) was cloned into the pGL2-Basic LUC vector and cotransfected with CMV-Empty vector (CMV-EV) or CMV-Foxf1. LUC assay shows FOXF1 transcriptionally activates the Cdh2 promoter. Data are representative of three independent experiments.

(C) Schematic drawings of the mouse Cadherin-11 (Cdh11) promoter. ChIP assays showed direct binding of FOXF1 to the indicated regions in the Cdh11 promoter. See also Figure S7 and Table S2.

(D) Schematic drawing of the pGL2-mCdh11-Luc construct containing the Cdh11 promoter region. The mouse Cdh11 promoter (−2,497/−137 bp) was cloned into the pGL2-Basic LUC vector and cotransfection with CMV-EV or CMV-Foxf1. LUC assay shows FOXF1 transcriptionally inhibits the Cdh11 promoter.

(E) Re-expression of CDH2 in FOXF1-deficient human CCL-210 lung fibroblasts decreased the invasion of fibroblasts through the basement membrane matrix (upper panels). CCL-210 lung fibroblasts were transfected with control siRNA, FOXF1 siRNA, control empty vector, or CDH2 OE, or cotransfected with siFOXF1 and CDH2 OE constructs. The efficiency of transfection is shown using qRT-PCR and western blot (bottom panels). Representative images and quantification of CCL-210 invasion are shown. Data presented as mean ± SEM.

(F) Depletion of CDH11 in FOXF1-deficient human lung fibroblasts decreased the invasion of fibroblasts through the basement membrane matrix (upper panels). CCL-210 fibroblasts were transfected with control siRNA, FOXF1 siRNA, or CDH11 siRNA or cotransfected with siFOXF1 and siCDH11. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. See also Figure S7.

(G) Schematic drawing shows loss of FOXF1 during myofibroblast differentiation is associated with the loss of CDH2 and gain of CDH11. FOXF1 directly regulates the CDH2-CDH11 cadherin switch by transcriptionally activating CDH2 and repressing CDH11. The cadherin switch is essential for myofibroblast differentiation, migration, and invasion.