Fig. 4.

Exercise training reduced POU2F1 by activating AMPK and downregulating C/EBPβ. (A) schematic of the binding site between C/EBPβ and the mouse POU2F1 promoter region and the structures of the reporter plasmids carrying the full POU2F1 promoter region (POU2F1 wild type) or the −1457 to −1448 bp site deletion fragment (POU2F1 binding site deletion); (B) HEK 293A cells were transfected with the wild-type or mutant plasmid and then treated with control plasmid or C/EBPβ overexpression plasmid, respectively. A dual-luciferase reporter assay was performed (n = 3); (C) ChIP analysis using anti-C/EBPβ antibody or IgG, soluble chromatin (500−600 bp in length) from CFs overexpressing C/EBPβ, and primers targeting the region spanning the C/EBPβ binding sites in the POU2F1 promoter; (D) ChIP analysis using anti-C/EBPβ antibody or IgG, soluble chromatin (500−600 bp in length) from mouse heart tissues, and primers targeting the region spanning the C/EBPβ binding sites in the POU2F1 promoter; (E) the expression of C/EBPβ was determined by qPCR; (F) the expression of POU2F1 was determined by qPCR; (G) representative images and quantitative analysis of the Western blot for (H) C/EBPβ and POU2F1, as well as (I) fibronectin and α-SMA; (J) the secretion of collagen I in the cell culture supernatant of CFs was determined by ELISA; (K) representative images and quantitative analysis of the Western blot for (L) p-AMPK to total AMPK and (M) C/EBPβ to GAPDH in the Ang II-induced cardiac fibrosis model; (N−Q) CFs were pretreated with compound C (1 μmol/L) for 0.5 h and treated with metformin (1 mmol/L) for 0.5 h. Then, Ang II (10⁻⁶ mol/L) was added to CFs for 48 h; (N) representative images and quantitative analysis of the relative protein expressions of (O) p-AMPK to total AMPK, (P) C/EBPβ to GAPDH, and (Q) POU2F1 to GAPDH in harvested CFs. (R) ChIP analysis using anti-C/EBPβ antibody or IgG, soluble chromatin (500−600 bp in length) from CFs treated with AngII and/or metformin, and primers targeting the region spanning the C/EBPβ binding sites in the POU2F1 promoter. Data are presented as mean ± SEM. n = 6 in (C−R). The t test was used in (C), (E), (F), and (H−J). A two-way analysis of variance followed by Tukey's post hoc test was used in (B), (L), and (M). A one-way analysis of variance was used in (O−R). * p < 0.05; ** p < 0.01; *** p < 0.001. Ad-Ctrl = control adenovirus; AMPK = AMP-activated protein kinase; Ang II = angiotensin II; bp = base pair; C/EBPβ = CCAAT enhancer-binding protein β; CFs = cardiac fibroblasts; ChIP = chromatin immunoprecipitation; ELISA = enzyme-linked immunosorbent assay; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; IgG = immunoglobulin G; ns = not significant; p-AMPK = phospho-AMP-activated protein kinase Thr 172; POU2F1 = POU domain, class 2, transcription factor 1; qPCR = quantitative polymerase chain reaction; Run = running; Sed = sedentary; SEM = standard error of mean.