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Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling.

Kuiken HJ, Egan DA, Laman H, Bernards R, Beijersbergen RL, Dirac AM - J. Cell. Mol. Med. (2012)

Bottom Line: Using cytoplasmic to nuclear translocation of the NF-κB transcription factor family member p65 as a read-out, we screened a synthetic siRNA library targeting enzymes involved in ubiquitin conjugation and de-conjugation for modifiers of regulatory ubiquitination events in NF-κB signalling.We identified F-box protein only 7 (FBXO7), a component of Skp, Cullin, F-box (SCF)-ubiquitin ligase complexes, as a negative regulator of NF-κB signalling.F-box protein only 7 binds to, and mediates ubiquitin conjugation to cIAP1 and TRAF2, resulting in decreased RIP1 ubiquitination and lowered NF-κB signalling activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

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FBXO7 regulates NF-κB signalling. (A and B) NF-κB target gene, A20 and IL8, activation determined by qRT PCR upon stimulation with TNFα in (A) U2OS and (B) immortalized primary human fibroblasts (BJET) transfected with siRNAs targeting FBXO7, TNF-R1, CYLD or a non-targeting (scrambled) control. Values are mean ± standard deviation of replicate measurements from a representative experiment. (C) NF-κB luciferase reporter assay in U2OS cells expressing the NF-κB luciferase reporter construct, a SV40-Renilla construct, and either empty vector (pcDNA3.1), Flag-tagged wild-type FBXO7 (WT), or Flag-tagged F-box deletion mutant FBXO7 (ΔF). Values represent ratio of Luciferase activity over Renilla control activity in the absence or presence of TNFα. *P < 0.05, values are mean ± standard deviation of four independent experiments. P-value computed from unpaired two-tailed t-test.
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fig02: FBXO7 regulates NF-κB signalling. (A and B) NF-κB target gene, A20 and IL8, activation determined by qRT PCR upon stimulation with TNFα in (A) U2OS and (B) immortalized primary human fibroblasts (BJET) transfected with siRNAs targeting FBXO7, TNF-R1, CYLD or a non-targeting (scrambled) control. Values are mean ± standard deviation of replicate measurements from a representative experiment. (C) NF-κB luciferase reporter assay in U2OS cells expressing the NF-κB luciferase reporter construct, a SV40-Renilla construct, and either empty vector (pcDNA3.1), Flag-tagged wild-type FBXO7 (WT), or Flag-tagged F-box deletion mutant FBXO7 (ΔF). Values represent ratio of Luciferase activity over Renilla control activity in the absence or presence of TNFα. *P < 0.05, values are mean ± standard deviation of four independent experiments. P-value computed from unpaired two-tailed t-test.

Mentions: Having established that FBXO7 inhibition leads to increased nuclear accumulation of p65, we next examined whether or not NF-κB target gene activation is also affected. The NF-κB target genes IL8, A20, IRF1 and STX11 were selected for these experiments based on micro-array studies in U2OS cells treated with TNFα for 1–2 hrs (data not shown). We measured the TNFα-induced activation of these NF-κB target genes by qRT PCR on RNA isolates of U2OS cells transfected with siRNAs targeting TNF-R1, CYLD or FBXO7 (Fig. 2A and S1). As expected, knockdown of TNF-R1 impairs the activation of these target genes. In contrast, knockdown of CYLD results in an increase of transcriptional activation of IL8, A20 and IRF1. Similarly to CYLD, knockdown of FBXO7, using two unique siRNAs, results in hyper-activation of IL8, A20, IRF1 and STX11. Importantly, we observed similar effects on target gene activation in an independent cell line, BJ primary fibroblasts immortalized by stable expression of human telomerase (hTERT). In these cells, suppression of FBXO7 expression also leads to increased transcriptional activation of IL8 and A20 upon TNFα stimulation (Fig. 2B).


Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling.

Kuiken HJ, Egan DA, Laman H, Bernards R, Beijersbergen RL, Dirac AM - J. Cell. Mol. Med. (2012)

FBXO7 regulates NF-κB signalling. (A and B) NF-κB target gene, A20 and IL8, activation determined by qRT PCR upon stimulation with TNFα in (A) U2OS and (B) immortalized primary human fibroblasts (BJET) transfected with siRNAs targeting FBXO7, TNF-R1, CYLD or a non-targeting (scrambled) control. Values are mean ± standard deviation of replicate measurements from a representative experiment. (C) NF-κB luciferase reporter assay in U2OS cells expressing the NF-κB luciferase reporter construct, a SV40-Renilla construct, and either empty vector (pcDNA3.1), Flag-tagged wild-type FBXO7 (WT), or Flag-tagged F-box deletion mutant FBXO7 (ΔF). Values represent ratio of Luciferase activity over Renilla control activity in the absence or presence of TNFα. *P < 0.05, values are mean ± standard deviation of four independent experiments. P-value computed from unpaired two-tailed t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822984&req=5

fig02: FBXO7 regulates NF-κB signalling. (A and B) NF-κB target gene, A20 and IL8, activation determined by qRT PCR upon stimulation with TNFα in (A) U2OS and (B) immortalized primary human fibroblasts (BJET) transfected with siRNAs targeting FBXO7, TNF-R1, CYLD or a non-targeting (scrambled) control. Values are mean ± standard deviation of replicate measurements from a representative experiment. (C) NF-κB luciferase reporter assay in U2OS cells expressing the NF-κB luciferase reporter construct, a SV40-Renilla construct, and either empty vector (pcDNA3.1), Flag-tagged wild-type FBXO7 (WT), or Flag-tagged F-box deletion mutant FBXO7 (ΔF). Values represent ratio of Luciferase activity over Renilla control activity in the absence or presence of TNFα. *P < 0.05, values are mean ± standard deviation of four independent experiments. P-value computed from unpaired two-tailed t-test.
Mentions: Having established that FBXO7 inhibition leads to increased nuclear accumulation of p65, we next examined whether or not NF-κB target gene activation is also affected. The NF-κB target genes IL8, A20, IRF1 and STX11 were selected for these experiments based on micro-array studies in U2OS cells treated with TNFα for 1–2 hrs (data not shown). We measured the TNFα-induced activation of these NF-κB target genes by qRT PCR on RNA isolates of U2OS cells transfected with siRNAs targeting TNF-R1, CYLD or FBXO7 (Fig. 2A and S1). As expected, knockdown of TNF-R1 impairs the activation of these target genes. In contrast, knockdown of CYLD results in an increase of transcriptional activation of IL8, A20 and IRF1. Similarly to CYLD, knockdown of FBXO7, using two unique siRNAs, results in hyper-activation of IL8, A20, IRF1 and STX11. Importantly, we observed similar effects on target gene activation in an independent cell line, BJ primary fibroblasts immortalized by stable expression of human telomerase (hTERT). In these cells, suppression of FBXO7 expression also leads to increased transcriptional activation of IL8 and A20 upon TNFα stimulation (Fig. 2B).

Bottom Line: Using cytoplasmic to nuclear translocation of the NF-κB transcription factor family member p65 as a read-out, we screened a synthetic siRNA library targeting enzymes involved in ubiquitin conjugation and de-conjugation for modifiers of regulatory ubiquitination events in NF-κB signalling.We identified F-box protein only 7 (FBXO7), a component of Skp, Cullin, F-box (SCF)-ubiquitin ligase complexes, as a negative regulator of NF-κB signalling.F-box protein only 7 binds to, and mediates ubiquitin conjugation to cIAP1 and TRAF2, resulting in decreased RIP1 ubiquitination and lowered NF-κB signalling activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Show MeSH
Related in: MedlinePlus