Fig. 4
Identification of KLF5-binding site to GDF15 promoter and effect of C5a stimulation, KLF5 overexpression or knockdown on KLF5 and GCN5 binding to the promoter. a Schematic drawing of GDF15 promoter region (−2078 to +103 nt). On the basis of five KLF5 response elements (RE1 to RE5) predicted by JASPAR, the reporter plasmids of GDF15-FL (−2078 to +103 nt), truncate 1 (−1682 to +103 nt), truncate 2 (−1039 to +103 nt), truncate 3 (−401 to +103 nt) and truncate 4 (−55 to +103 nt) promoters were constructed. b, c A549 cells were transfected with the GDF15-FL or different truncated GDF15 promoters and then treated with C5a stimulation for 3 h (b) or pIRES2-KLF5 transfection for 48 h (c). Luciferase reporter analysis showed that the GDF15 promoter activity was markedly increased upon C5a or KLF5 overexpression (**P < 0.01 vs. without C5a or pIRES2-EGFP), except for truncate 4 group, and the promoter activity in truncate 4 group was dramatically lower than that in GDF15-FL, truncate 1, 2, and 3 groups (△△P < 0.01). d A549 cells were co-transfected with the above-mentioned GDF15 promoter plasmids and shKLF5 or shCTR plasmids. The GDF15 promoter activity was suppressed when KLF5 was silenced, even in the present of C5a stimulation (**P < 0.01), apart from truncate 4 group, and the activity in truncate 4 group reduced significantly compared with GDF15-FL, truncate 1, 2, and 3 groups (△△P < 0.01). e A549 cells were treated with C5a, and then ChIP assay was performed with KLF5 antibody. The purified DNA was amplified by PCR for GDF15 promoter regions of −234 to −76 nt (containing RE4) and −103 to +58 nt (containing RE5). It showed that C5a greatly increased the level of KLF5 binding to GDF15 promoter (−103 to +58 nt). f A549 cells were transfected with the GDF15-FL or GDF15-RE5-Mut reporter plasmid and stimulated by C5a for 3 h. Luciferase assay showed the mutation of RE5 could markedly inhibit the C5a-induced activation of GDF15 promoter. g ChIP assay using anti-KLF5 and real-time PCR revealed that the content of KLF5 binding to the region of GDF15 promoter elevated in both C5a and pIRES2-KLF5 groups (**P < 0.01 vs. DMEM; △△P < 0.01 vs. pIRES2-EGFP), and reduced in shKLF5 + C5a group (##P < 0.01 vs. shCTR + C5a). h A549 cells were stimulated by C5a for 3 h. Anti-KLF5 and GCN5 abs were used to perform ChIP and Re-ChIP assays. It showed that the KLF5 could bind to GDF15 promoter (−103 to +58 nt) together with GCN5 (**P < 0.01 vs. IgG). i ChIP performed by using the anti-GCN5 ab exhibited that the binding of GCN5 to GDF15 promoter (−103 to +58 nt) increased with C5a stimulation (**P < 0.01 vs. DMEM), but diminished by KLF5 interference (△△P < 0.01 vs. shCTR + C5a). Representative photographs are displayed. All data represent means ± S.E.M. of three independent experiments
