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

Verstrepen L, Adib-Conquy M, Kreike M, Carpentier I, Adrie C, Cavaillon JM, Beyaert R - J. Cell. Mol. Med. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, Ghent, Belgium.

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.

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Related in: MedlinePlus

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.
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fig01: 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.

Mentions: ABIN-3 expression has previously been described as an LPS-inducible gene in the monocytic cell line THP-1 and in bone marrow-derived macrophages [9, 11]. To further analyse the inducible expression of ABIN-3 in other cell lines and cell types, we analysed ABIN-3 mRNA and protein expression in the mono-cytic cell lines THP-1 and U937s, the T cell line Jurkat, the hepatoma cell line HepG2 and the cervix carcinoma cell line HeLa. Cells were treated with LPS or TNF for 3 hrs, after which mRNA was isolated and analysed for ABIN-3 expression by semi-quantitative RT-PCR (Fig. 1A). Basal ABIN-3 mRNA expression could already be observed in THP-1 and HeLa cells, and to a lesser extent in Jurkat and HepG2 cells. LPS treatment up-regulated ABIN-3 mRNA expression in THP-1 and U937s cells. Similarly, also TNF increased ABIN-3 expression in these cells, as well as in HepG2 and HeLa cells. No up-regulation of ABIN-3 mRNA could be observed in Jurkat cells. Interestingly, ABIN-3 protein expression could only be detected in LPS stimulated THP-1 and U937s cells, and in TNF stimulated HeLa cells (Fig. 1B). These data demonstrate that ABIN-3 expression is not restricted to LPS stimulated myeloid cells, but also occurs in other cell types in response to TNF. Moreover, they show that ABIN-3 expression is regulated at the transcriptional and translational level.


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

Verstrepen L, Adib-Conquy M, Kreike M, Carpentier I, Adrie C, Cavaillon JM, Beyaert R - J. Cell. Mol. Med. (2007)

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.
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Related In: Results  -  Collection

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fig01: 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.
Mentions: ABIN-3 expression has previously been described as an LPS-inducible gene in the monocytic cell line THP-1 and in bone marrow-derived macrophages [9, 11]. To further analyse the inducible expression of ABIN-3 in other cell lines and cell types, we analysed ABIN-3 mRNA and protein expression in the mono-cytic cell lines THP-1 and U937s, the T cell line Jurkat, the hepatoma cell line HepG2 and the cervix carcinoma cell line HeLa. Cells were treated with LPS or TNF for 3 hrs, after which mRNA was isolated and analysed for ABIN-3 expression by semi-quantitative RT-PCR (Fig. 1A). Basal ABIN-3 mRNA expression could already be observed in THP-1 and HeLa cells, and to a lesser extent in Jurkat and HepG2 cells. LPS treatment up-regulated ABIN-3 mRNA expression in THP-1 and U937s cells. Similarly, also TNF increased ABIN-3 expression in these cells, as well as in HepG2 and HeLa cells. No up-regulation of ABIN-3 mRNA could be observed in Jurkat cells. Interestingly, ABIN-3 protein expression could only be detected in LPS stimulated THP-1 and U937s cells, and in TNF stimulated HeLa cells (Fig. 1B). These data demonstrate that ABIN-3 expression is not restricted to LPS stimulated myeloid cells, but also occurs in other cell types in response to TNF. Moreover, they show that ABIN-3 expression is regulated at the transcriptional and translational level.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, Ghent, Belgium.

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.

Show MeSH
Related in: MedlinePlus