Pre-B-cell Colony-enhancing Factor (PBEF/Visfatin) Gene Expression is Modulated by NF-κB and AP-1 in Human Amniotic Epithelial Cells
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Fig. 1
WISH (n=6) and primary amniotic epithelial cells (n=5) were stimulated with IL-1β (1 and 10 ng/ml) for 4 h, PBEF (A and C) and IL-8 (B and D) gene expression were quantitated by real-time PCR. IL-1β treatment caused a significant up-regulation of PBEF at both concentrations in the WISH cells but only at 1 ng/ml in the primary amniotic epithelial cells. However, the IL-8 gene was up-regulated in both cell types at both concentrations used. *p<0.05, **p<0.005, ***p<0.001. Data are shown relative to 18s and error bars are standard error of the mean.
Fig. 2
Stimulation of the amniotic epithelial cell line (WISH) with IL-1β (1 ng/ml) caused the nuclear translocation of NF-κB. Fluorescent labeling of WISH cells at time zero showed cytosolic labeling of the NF-κB subunit, p65 (A). After 1 h of stimulation with IL-1β (1 ng/ml) immunolabeling of p65 showed its nuclear location (B). Magnification ×600. An example experiment shows the percentage of cells with nuclear p65 label after stimulation with IL-1β (1 ng/ml) for 4 h (C). An example of a Western blot of cytosolic (Cy) and nuclear (Nu) cell protein fractions with an antibody to p65 at time zero and after stimulation with IL-1β (1 ng/ml) for 1 h (D).
Fig. 3
The stimulation of primary amniotic epithelial cells with IL-1β (1 ng/ml), led to the nuclear translocation of NF-κB. An example of a Western blot probed with antibody to the NF-κB subunit p65, showing the time-course (A), below this the blot is re-probed with a nuclear protein loading control (HP-1α). Quantitation (n=5 experiments) (B) show significant increases after 15 and 30 min of stimulation compared to the control. Stimulation with IL-1β also increased the phosphorylation of c-Jun, shown in the Western blot (c-JunP) and its loading control (c-Jun) (C). Beneath is the quantitation (n=5), with a significant increase by 15 min, continuing until 120 min before declining (D). Stimulation of c-Fos phosphorylation is shown in the Western blot for c-FosP and its loading control c-Fos (E). The quantitation (n=5) beneath shows a significant increase at 30 min, continuing until 120 min before decreasing (F). All data shown are relative to the T0 control and error bars are standard error of the mean. *p<0.05, **p<0.005.
Fig. 4
A representative immunolabeling experiment showing percentage of WISH cells containing nuclear p65 label (A). Cells were treated with IL-1β (1 ng/ml) for 30 min with either SN50 (50 μM) or curcumin (50 μM) and immunolabeled with anti-p65. Cells were positive if they showed nuclear label. Western blots of primary amniotic epithelial cells were similarly treated with IL-1β (1 ng/ml) with either SN50 (50 μM), curcumin (50 μM) or JNK inhibitor for 30 min. Representative blots are shown above each for p65 (B), phosphorylated c-Jun (C) or phosphorylated c-Fos (D) and addition of IL-1β with or without inhibitor is indicated by + or −.
Fig. 5
WISH cells were stimulated for 4 h with IL-1β (1 ng/ml) with or without SN50 50 μM (A), a JNK inhibitor 20 μM (B) or curcumin 50 μM (C). RNA was isolated and quantitative real-time PCR used to measure PBEF gene expression. Results were normalized to 18s and expressed relative to control (no treatment), error bars are ±standard error of the mean, *p<0.05, **p<0.005, ***p<0.001. Treatment with IL-1β significantly increased PBEF gene expression compared to media alone (A–C). Addition of SN50 (n=8) significantly inhibited the up-regulation of PBEF by IL-1β. Results with the JNK inhibitor (n=7) also caused an inhibition of PBEF up-regulation, not reaching significance. The addition of curcumin (n=7) significantly inhibited PBEF gene expression when increased by IL-1β. Primary amniotic epithelial cells were used in similar experiments (n=3 different patients) (D). IL-1β increased PBEF gene expression, the same pattern of inhibition as in WISH cells was caused by the addition of the same inhibitors together with IL-1β. SN50, JNK and curcumin all inhibited the increase in PBEF and the combination of SN50 and JNK had an additive effect inhibiting PBEF below baseline levels (D).
Fig. 6
RNA from the experiments in Fig. 5 was used for quantitative real-time PCR for IL-8 gene expression. Results were normalized to 18s expressed relative to control (no treatment), error bars are ±standard error of the mean. *p<0.05, **p<0.005. Treatment with IL-1β significantly increased IL-8 gene expression compared to media alone (A–C). Addition of SN50 inhibited this but was not significant (A). The JNK inhibitor also inhibited IL-8 up-regulation but again this did not reach significance (B). However, a significant inhibition was caused by the addition of curcumin (C) with primary amniotic epithelial cells. IL-1β significantly up-regulated IL-8 gene expression and the same pattern of inhibition was caused by the addition of the inhibitors when added with IL-1β (D). SN50, JNK and curcumin all inhibited the up-regulation of IL-8.
Abstract
A localized intrauterine inflammatory response is often associated with the initiation of normal human parturition, whereas infection causes a similar but more florid response initiating preterm labor. Pre-B-cell colony-enhancing factor (PBEF) is expressed in the human fetal membranes and is up-regulated by labor, severe infection and inflammatory stimuli. The aim of this study was to determine the involvement of the transcription factors NF-κB and AP-1 in the response of PBEF to an inflammatory stimulus and compare it with IL-8. The results showed that this treatment of amniotic epithelial-like cells (WISH) and primary amniotic epithelial cells increased expression of PBEF and IL-8, but IL-8 responded 100-fold more than PBEF. IL-1β treatment together with a panel of NF-κB and AP-1 inhibitors demonstrated the involvement of these transcription factors in the up-regulation of PBEF. These data show that an inflammatory stimulus in the fetal membranes inducing NF-κB and AP-1 would up-regulate PBEF as well as IL-8.
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