Figure 6. KLF5 transactivates the Igf1 promoter.

(A) KLF5 knockdown reduced Igf1 expression in cardiac fibroblasts. KLF5 was knocked down as shown in Figure 5A. *P < 0.01 versus siCntrl. (B) Fibroblast-selective expression of Igf1. Igf1 mRNA levels in cultured cardiac fibroblasts were normalized to those of 18s rRNA and then further normalized with respect to those in cardiomyocytes. *P < 0.01 versus cardiomyocytes. (C) Cardiac expression of Klf5 and Igf1 mRNA after LI-TAC in Klf5^fl/fl and Klf5^fl/fl;Postn-Cre mice. Expression levels were normalized to those of 18s rRNA and then further normalized with respect to those in the hearts before TAC. (D) Reporter analysis of KLF5-depedent transactivation of the Igf1 promoter. Luciferase reporter constructs driven by the wild-type Igf1 promoter or a mutant promoter in which the potential KLF-binding site was mutated were cotransfected with either empty vector or a vector harboring Klf5 or Klf15. Data are representative of 3 independent experiments. (E) ChIP assays of KLF5 binding to the Igf1 and Pdgfa promoters. An intronic region of Igf1 that does not contain a KLF-binding motif served as a negative control. (F) Effects of neutralizing IGF-1 on the cardiotrophic activity of fibroblast-conditioned medium. An antibody against IGF-1 (30 μg/ml) or normal IgG was added to the conditioned medium, after which the effect of the medium on cardiomyocyte surface area was analyzed. *P < 0.01 versus cells treated with SFM; ^#P < 0.01 versus cells treated with fibroblast-conditioned medium.