Canonical NF-κB p65, but Not p105, Contributes to IL-1β-Induced IL-8 Expression in Cardiac Fibroblasts

Abstract

Cardiac fibroblasts participate in the inflammatory process of heart diseases as sentinel cells of the cardiac tissue. In this study, we investigated the effect of the proinflammatory cytokine, interleukin 1β (IL-1β), on the expression of interleukin 8 (IL-8), which contributes to the induction of innate immunity via the activation and recruitment of innate immune cells, such as neutrophils, to the site of inflammation in canine cardiac fibroblasts. IL-1β mediates IL-8 mRNA expression and protein release in a dose- and time-dependent manner. The IL-β-mediated IL-8 protein release and mRNA expression were inhibited by 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide, an inhibitor of the transcription factor, nuclear factor (NF)-κB. In cells treated with IL-1β, NF-κB p65 and p105 were transiently phosphorylated, indicating the activation of NF-κB. However, IL-1β failed to induce IL-8 mRNA expression in the cells transfected with p65 small interfering RNA (siRNA), but not in those transfected with p105 siRNA. These observations suggest that IL-1β induces IL-8 expression via the activation of NF-κB p65 in canine cardiac fibroblasts.

Keywords: NF-κB p65; cardiac fibroblasts; inflammation; interleukin 1β; interleukin 8.

Figure 1

IL-1β-mediated IL-8 release and IL-8 mRNA expression in canine cardiac fibroblasts. Time-dependent increase in IL-8 protein release (A) and IL-8 mRNA expression levels (C) in fibroblasts treated with (closed circle) or without (open circle) canine recombinant IL-1β (100 pM). Dose-dependent IL-8 protein release (B) and IL-8 mRNA expression levels (D) in fibroblasts treated with the indicated concentrations of IL-1β for 24h. HRPT1 was used as an internal standard and the expression levels of IL-8 mRNA in IL-1β-stimulated fibroblasts were compared with the expression at 0h. Results have been represented as mean ± standard error (SE) from biological triplicates. *P <0.05.

Figure 2

Effect of the NF-κB inhibitor TPCA-1 on IL-1β-mediated IL-8 mRNA expression. Canine cardiac fibroblasts were pretreated with or without TPCA-1 (10 µM) for 1 h and subsequently stimulated with or without IL-1β (100 pM) for 24 h. After stimulation, IL-8 mRNA expression levels (A) and the release of IL-8 (B) were determined. HRPT1 was used as an internal standard and the expression levels of IL-8 mRNA in IL-1β-stimulated fibroblasts were compared with the expression at 0 h. Results have been represented as mean ± standard error (SE) from biological triplicates. *P <0.05.

Figure 3

IL-1β-mediated activation of NF-κB. (A) Representative western blotting results of levels of total IκBα (t-IκBα), phosphorylated p65 (p-p65), total p65 (t-p65), phosphorylated p105 (p-p105), and total p105 (t-p105) in canine cardiac fibroblasts treated with IL-1β (100 pM). β-actin was used as an internal standard. Time-dependent changes of levels of t-IκBα, p-p65 and p-p105 were observed. Relative levels of t-IκBα (B), [p-p65]/[t-p65] (C), and [p-p105]/[t-p105] (D) in IL-1β-stimulated fibroblasts compared to the levels at 0h. Results have been represented as mean ± SE from biological triplicates. *P <0.05.

Figure 4

Effect of the NF-κB inhibitor TPCA-1 on IL-1β mediated phosphorylation of p65 and p105. Canine cardiac fibroblasts were pretreated with or without TPCA-1 (10 µM) for 1 h and stimulated with IL-1β (100 pM) for 15 min. Representative western blotting results of inhibitory effect of TPCA-1 on IL-1β-mediated phosphorylation of p65 (A) and p105 (C), and relative levels of [p-65]/[t-65] (B) and [p-105]/[t-105] (D) as compared to those without the inhibitor and IL-1β. Results have been represented as mean ± SE from biological triplicates. *P <0.05.

Figure 5

Transfection with p65 siRNA attenuated IL-1β-mediated IL-8 mRNA expression in canine cardiac fibroblasts but not that with p105 siRNA. In canine cardiac fibroblasts transfected with p65, p105, and scrambled siRNAs, expression of t-p65, t-105, and β-actin was detected by western blotting. The expression of p65 or p105 was reduced in fibroblasts transfected with p65 or p105 siRNA, respectively, but not in fibroblasts transfected with scrambled siRNA. β-actin was used as an internal standard. Representative results (A) and relative density of protein expression of t-p65 (B) or t-p105 (C) in siRNA-transfected fibroblasts compared with those in scrambled siRNA-transfected fibroblasts are depicted. (D) Cardiac fibroblasts transfected with p65, p105 and scrambled siRNAs were incubated with or without IL-1β (100 pM) for 24 h After the incubation, IL-8 mRNA expression was determined. HRPT1 was used as an internal standard. Transfection with p65 siRNA resulted in reduction of IL-1β-mediated IL-8 mRNA expression, while p105 and scrambled siRNA-transfection did not. Results have been represented as mean ± standard error (SE) from biological triplicates. *P <0.05; N.S, Not Significant.