Krüppel-Like Factor 5 Mediates Proinflammatory Cytokine Expression in Lipopolysaccharide-Induced Acute Lung Injury through Upregulation of Nuclear Factor-κB Phosphorylation In Vitro and In Vivo
Figure 4
Effects ofKrüppel-like factor 5 (KLF5) overexpression or silencing on p65 phosphorylation and nuclear factor-kappaB (NF-κB) activity. (a) Cells from the HBEC line were pretreated with the pKLF5 plasmid or transfected with KLF5 small interfering RNA (siRNA) for 48 h, and KLF5, p65, phospho-p65 (Ser276), and phospho-p65 (Ser536) protein expression were measured using western blotting. (b) NF-κB activity was measured using an electrophoretic mobility shift assay, and (c) the proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1β, and IL-6 were evaluated using western blotting. (d) Cells from the HBEC line were pretreated with N-acetylcysteine (NAC; 10 mM) or pyrrolidine dithiocarbamate (PDTC; 100 μM) for 1 h and then treated with lipopolysaccharide (LPS; 5 μg/mL) for 1 h. KLF5 protein expression was evaluated using western blotting. Silencing of KLF5 messenger RNA (mRNA) expression reduced LPS-induced p65 accumulation in the nucleus. (a) Compared with that in the control group, cells treated with LPS alone, or cells transfected with KLF5-specific small interfering RNA (siRNA), NF-κB binding activity in cells transfected with the KLF5 plasmid increased in response to LPS. (b) Compared with those in the control group, cells treated with LPS alone, or cells transfected with KLF5-specific siRNA, levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 in cells transfected with the KLF5 plasmid were increased. (c) Cells pretreated with the NF-κB inhibitor PDTC (100 μM) for 1 h exhibited no reduction in KLF5 protein expression after LPS exposure. (d) Data are presented as means ± SEM from 3 independent experiments. Each bar graph shows summarized data (mean ± SEM) from 3 separate densitometry experiments after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; an internal control for nuclear protein). versus medium alone or control condition.
