Expression of the NF-kappaB inhibitor ABIN-3 in response to TNF and toll-like receptor 4 stimulation is itself regulated by NF-kappaB

Abstract

Although the nuclear factor-kappaB (NF-kappaB)-dependent gene expression is critical to the induction of an efficient immune response to infection or tissue injury, excessive or prolonged NF-kappaB signalling can contribute to the development of several inflammatory diseases. Therefore, the NF-kappaB signal transduction pathway is tightly regulated by several intracellular proteins. We have previously identified A20-binding inhibitor of NF-kappaB activation (ABIN)-3 as an lipopolysaccharide (LPS)-inducible protein in monocytes that negatively regulates NF-B activation in response to tumour necrosis factor (TNF) and LPS. Here we report that ABIN-3 expression is also up-regulated upon TNF treatment of monocytes and other non-myeloid cell types. We also found a significantly enhanced expression of ABIN-3 in monocytes of sepsis patients, which is restored to control levels by corticotherapy. To further understand the transcriptional regulation of ABIN-3 expression, we isolated the human ABIN-3 promoter and investigated its activation in response to TNF and LPS. This revealed that the LPS- and TNF-inducible expression of ABIN-3 is dependent on the binding of NF-kappaB to a specific B site in the ABIN-3 promoter. Altogether, these data indicate an important role for NF-kappaB-dependent gene expression of ABIN-3 in the negative feedback regulation of TNF receptor and toll-like receptor 4 induced NF-kappaB activation.

1

ABIN-3 expression is up-regulated in response to LPS and TNF.(A) Semi-quantitative RT-PCR of ABIN-3 mRNA expression in different cell lines. RNA was isolated from the indicated cell lines that were either left untreated or stimulated for 3 hrs with 100 ng/ml LPS or 1000 IU/ml TNF. Semi-quantitative RT-PCR was performed using ABIN-3 specific primers to amplify a 700 bp fragment of the ABIN-3 open-reading frame. β-actin was used as a control.(B) Western blotting for ABIN-3 protein expression in different cell lines. Cells were either left untreated or stimulated for 6 or 18 hrs with LPS or TNF as indicated. Cell extracts were separated by SDS-PAGE and analysed by Western blotting and immunode-tection with anti-ABIN-3 polyclonal antibodies. β-actin expression was used as a control for equal loading.(C) Real-time quantitative PCR of ABIN-3 expression in monocytes from sepsis patients. Monocytes were selected by adherence from PBMC of healthy donors (n= 12) and septic shock patients (n= 6) before and 24 hrs after low-dose HC therapy. Total RNA was isolated and ABIN-3, SIGIRR and MyD88s expression were analysed by real time quantitative PCR using specific primers. All results were normalized with respect to the expression of GAPDH.*P<0.01 patients versus healthy controls (Anova), P<0.05 patients before HC versus patients after HC (Wilcoxon signed-rank test).(D) Effect of HC on LPS-induced ABIN-3 mRNA expression in primary human monocytes. Monocytes were selected by adherence from PBMC of healthy donors. Cells were stimulated with 100 ng/ml LPS for 20 hrs in the presence or the absence of 100 μM HC, which was given 2 hrs after LPS. Total RNA was isolated and ABIN-3 mRNA expression was analysed by real-time PCR. Results were normalized with respect to the expression of GAPDH and represent the mean ± SD of three experiments performed on cells from different donors.(E) Protein expression of ABIN-3 in monocytes isolated from healthy donors. Cells were stimulated with 100 ng/ml LPS for 20 hrs in the presence or the absence of 100 M HC, which was given at the same time as LPS. Cell extracts were separated by SDS-PAGE and analysed by Western blotting and immunodetection with anti-ABIN-3 polyclonal antibodies. β-actin expression was used as a control for equal loading.

2

NF-κB inhibitors prevent the LPS-induced expression of ABIN-3. (A) Semi-quantitative RT-PCR of ABIN-3 mRNA. RNA was isolated from THP-1 cells that were either left untreated or stimulated for 3 hrs with 100 ng/ml LPS in the presence or the absence of 50 μM sc-514, 2.5 μM MG-132 or the solvent control DMSO, which were given 1 hr before LPS. Semi-quantitative RT-PCR was performed using ABIN-3 specific primers to amplify a 700 bp fragment of the ABIN-3 open-reading frame. β-actin was used as a control. (B) Western blotting for ABIN-3 protein expression. THP-1 cells that were either left untreated or stimulated for 6 hrs with 100 ng/ml LPS in the presence or the absence of 50 μM sc-514, 2.5 μM MG-132 or the solvent control DMSO, which were given 1 hr before LPS. Cell extracts were separated by SDS-PAGE and analysed by Western Blotting and immunodetection with anti-ABIN-3 polyclonal antibodies. β-actin expression was used as a control for equal loading.

3

Nucleotide sequence of the ABIN-3 promoter.2130 bp fragment of the ABIN-3 promoter still containing at its 3′ end 36 bp of the ABIN-3 cDNA (shown in italics). Binding sites for transcription factors were predicted using the MatInspector program. Only a selection of putative binding sites is shown. The putative κB element is shown in bold.

4

NF-κB binds to the κB site in the ABIN-3 promoter.(A) THP-1 cells were stimulated for the indicated times with 100 ng/ml LPS or 1000 IU/ml TNF. Nuclear extracts were prepared and analysed by electrophoretic mobility shift assay using ³²P-labelled WT-κB or mut-κB probes corresponding to the κB site in the ABIN-3 promoter.(B) Binding of p65 NF-κB to the WT-κB probe was demonstrated by a supershift upon pre-incubation of the nuclear extract (obtained from cells treated for 60 min.with LPS) with anti-p65 NF-κB antibodies. The arrow indicates the location of the supershifted NF-κB complex.

5

TNF, TLR4 and p65 induce the NF-κB-dependent activation of the ABIN-3 promoter. The Luc reporter vector pXPΔ2 containing either no insert, the WT-κB ABIN-3 promoter fragment or the mut-κB ABIN-3 promoter fragment was transiently transfected in HEK293T cells together with pAct gal. NF-κB was activated by treating the cells for 6 hrs with 1000 IU/ml TNF or by co-expression of TLR4 or p65 as indicated. In (C), cells were also transfected with expression vectors for ABIN-3, A20 or IKKβ-K44A as indicated. Luc activity and Gal activity in cell lysates were assayed 24 hrs after transfection and values are plotted as Luc/Gal to adjust for differences in transfection efficiency. Each error bar represents the mean ±SD of two samples. Results are representative for at least two independent experiments.‘/’: non-stimulated.

6

Hydrocortisone inhibits LPS-induced activation of the ABIN-3 promoter in macrophages. The Luc reporter vector pXP2Δ2 containing either no insert or the WT-κB ABIN-3 promoter fragment was transiently transfected in RAW264.7 cells together with pAct βgal. Cells were either left untreated or stimulated for 6 hrs with 1 μg/ml LPS in the presence or absence of 100 μM HC that was given 2 hrs prior to LPS. Luc activity and Gal activity in cell lysates were assayed 24 hrs after transfection and values are plotted as Luc/Gal to adjust for differences in transfection efficiency. Each error bar represents the mean ± SD of two samples. Results are representative for at least two independent experiments.‘/’: non-stimulated.