Figure 1.
Klf4 expression is regulated in hearts in response to Ang II infusion.
A, Klf mRNA levels in C57BL/6J wild-type (WT) hearts after 7 days of saline or Ang II infusion (1500 ng.kg−1.min−1). mRNA levels were assessed by quantitative real-time PCR (qPCR) and normalized to β-tubulin. Klf12 and Klf14 were not detectable in hearts. Klf17 was not detectable in saline-infused hearts (n = 3). B, qPCR of Klf4 mRNA levels in WT hearts after 3 and 5 days of Ang II infusion (n = 3). Data are the mean ± SEM. *P<0.05 vs. saline-infused control. C, Klf mRNA levels in cardiac fibroblasts after 2 hrs of stimulation with PBS control or Ang II (1 μmol/L). Klf14 and Klf17 were not detectable in cardiac fibroblasts (n = 3). D, Western blot of Klf4 protein levels after Ang II treatment. GAPDH was the loading control. Data are the mean ± SEM. *P<0.05, **P<0.01 vs. PBS control.
Figure 2.
Elevated Klf4 promotes Ang II-induced fibroblast differentiation and collagen synthesis.
Cardiac fibroblasts were exposed to co-infection of Ad-tTA and Ad-Klf4 for 2 hrs. After the viruses were washed off, infected cells were further incubated for the indicated time in the presence or absence of Tet (tetracycline, 0.1 μg/ml) for 24 hrs, then the cells were treated with Ang II (1 μmol/L). A, immunofluorescence analysis of myofibroblast differentiation by staining with a α-smooth muscle actin (α-SMA) antibody after Ang II treatment for 0, 24 or 48 hrs. Scale bars: 10 μm. B, Quantitative analyses of α-SMA positive cells are presented (n = 4). C, α-SMA mRNA levels were assessed by quantitative real-time PCR (qPCR) after Ang II treatment for 0, 6 or 12 hrs and normalized to β-tubulin (n = 4). D, α-SMA protein levels were assessed by Western blot after Ang II treatment for 0, 24 or 48 hrs. GAPDH was a loading control. E, Level of quantification ofα-SMA as a ratio of GAPDH in densitometric units was presented. n = 4. F, Col1α1, Col1α2, Col3α1 mRNA levels were assessed by quantitative real-time PCR (qPCR) after Ang II treatment for 0, 6 or 12 hrs and normalized to β-tubulin. n = 4. Data are the mean ± SEM. *P<0.05, **P<0.01, ***P<0.01 vs. control. #P<0.05, ##P<0.01 vs. control and Ang II treatment for 0 hr.
Figure 3.
siKlf4 weakens Ang II-induced fibroblast differentiation and collagen synthesis.
The effect of Klf4 depends on cytokine secretion. A, semiquantitative PCR of the mRNA levels of Klf4 showed siRNA-mediated knockdown in cardiac fibroblasts. B, Klf4 mRNA levels were assessed by quantitative real-time PCR (qPCR) after siKlf4 transfection (n = 3). C, α-SMA mRNA levels were assessed by quantitative real-time PCR (qPCR) after Ang II treatment for 0, 6 or 12 hrs in siRNA transfected cardiac fibroblasts and normalized to β-tubulin (n = 3). Data are the mean ± SEM. *P<0.05, **P<0.01 vs. scrambled siRNA. D, cultured cardiac fibroblasts were incubated with supernatants collected from cultured cardiac fibroblasts that overexpressed Klf4 or control. Immunofluorescence analysis was performed after staining with α-SMA antibody. E, quantitative analyses of α-SMA positive cells are presented (n = 4). Data are the mean ± SEM. *P<0.05 vs. control supernatant.
Figure 4.
Klf4 controls Ang II-induced TGF-β1 production.
A, Klf4 and TGF-β1 mRNA levels were assessed by qPCR in Ang II-treated cardiac fibroblasts and were normalized to β-tubulin (n = 3). B, Klf4 and TGF-β1 protein levels were assessed by Western blot in Ang II-treated cardiac fibroblasts. GAPDH was the loading control (n = 3). Data are the mean ± SEM. *P<0.05, **P<0.01 vs. Ang II treatment for 0 hr. C-F, cardiac fibroblasts were co-infected with Ad-tTA and Ad-Klf4 and treated with Ang II. C, TGF-β1 mRNA levels were assessed by qPCR after Ang II treatment for 0, 6 or 12 hrs and normalized to β-tubulin (n = 4). D, TGF-β1 and phosphorylated Smad3 (p-Smad3) protein levels were assessed by Western blot after Ang II treatment for 0, 24 or 48 hrs. E, level of quantification of TGF-β1 and p-SMAD3 as a ratio of GAPDH in densitometric units was presented. n = 4. F, ELISA was used to analyze active TGF-β1 levels in the supernatant after Ang II treatment for 0, 24 or 48 hrs (n = 5). Data are the mean ± SEM. *P<0.05, **P<0.01 vs. control. #P<0.05, ##P<0.01 vs. control and Ang II treatment for 0 hr.
Figure 5.
Ang II-induced TGF-β1 production is partly dependent on Klf4.
A–C, cardiac fibroblasts were transfected with siKlf4 and treated with Ang II. A, Western blot showed siRNA-mediated knockdown of Klf4 and TGF-β1 protein levels in cardiac fibroblasts. B, TGF-β1 mRNA levels were assessed by qPCR after Ang II treatment for 0, 6 or 12 hrs (n = 3). C, ELISA was used to analyze active TGF-β1 levels in the supernatant of Klf4- or scrambled-siRNA transfected cardiac fibroblasts after Ang II treatment for 0, 24 or 48 hrs (n = 3). *P<0.05 vs. scrambled siRNA. D–F, adult cardiac fibroblasts were isolated from mice carrying LoxP floxed Klf4 alleles. The cells were infected with Ad-Cre for Klf4 deletion, and infected WT cells were served as the negative control. Ad-Cre, Ad-tTA and Ad-Klf4 were co-infected to reintroduce Klf4 expression. D, 2 days after adenovirus infection, the cells were analyzed for Klf4 protein expression by Western blot. E, TGF-β1 mRNA levels were assessed by qPCR after Ang II treatment for 0 or 6 hrs in Klf4-deleted and Klf4-reintroduced fibroblasts and were normalized to β-tubulin (n = 3). Data are the mean ± SEM. *P<0.05 vs. Ad-Cre infected WT control; #P<0.05, ##P<0.01 vs. Ad-Cre infected Klf4-floxed fibroblasts. F, TGF-β1 protein levels were assessed by Western blot after Ang II treatment for 24 hrs.
Figure 6.
Klf4 transactivates the TGF-β1 promoter.
A, ChIP assays of Klf4 binding to the −184∼−180-bp site (Klf BS1) and −45∼−41-bp site (Klf BS1) of the TGF-β1 promoter. A non-target region (GAPDH exon 2) was used as a negative control. B, normalization of each ChIP DNA fraction to the input DNA fraction using densitometric units (n = 3) was shown. Data are the mean ± SEM. *P<0.05, **P<0.01 vs. Ang II untreated and Klf4 non-overexpression. ##P<0.01 vs. Ang II treatment. C, pGL3-luciferase reporter driven by the wild-type TGF-β1 promoter or mutant (at −184∼−180-bp and −45∼−41-bp promoter sites) in which the potential Klf4-binding site was constructed. D, promoter-reporter analysis for Klf4-depedent transactivation of the TGF-β1. The pGL3-TGF-β1 WT or mutant vector and CMV-renilla luciferase vector were cotransfected. Luciferase activation driven by the wild-type TGF-β1 promoter or mutant promoter and were normalized to renilla luciferase (n = 4). Data are the mean ± SEM. E-F, effects of neutralizing TGF-β1 on the cardiac fibroblast differentiation activity. An antibody against TGF-β1 (1 μg/ml) was added to the conditional medium. E, the effects of Klf4-dependent α-SMA mRNA expression was assessed by qPCR after Ang II treatment for 0 or 6 hrs (n = 4). Data are the mean ± SEM. *P<0.05, **P<0.01 vs. Klf4 non-overexpression; #P<0.05, ##P<0.01 vs. Klf4 overexpression. F, levels of α-SMA and p-Smad3 were assessed by Western blot after Ang II treatment for 24 hrs.