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JMJD8 is a positive regulator of TNF-induced NF- κ B signaling

View Article: PubMed Central - PubMed

ABSTRACT

TNF-induced signaling mediates pleiotropic biological consequences including inflammation, immunity, cell proliferation and apoptosis. Misregulation of TNF signaling has been attributed as a major cause of chronic inflammatory diseases and cancer. Jumonji domain-containing protein 8 (JMJD8) belongs to the JmjC family. However, only part of the family members has been described as hydroxylase enzymes that function as histone demethylases. Here, we report that JMJD8 positively regulates TNF-induced NF-κB signaling. Silencing the expression of JMJD8 using RNA interference (RNAi) greatly suppresses TNF-induced expression of several NF-κB-dependent genes. Furthermore, knockdown of JMJD8 expression reduces RIP ubiquitination, IKK kinase activity, delays IκBα degradation and subsequently blocks nuclear translocation of p65. In addition, JMJD8 deficiency enhances TNF-induced apoptosis. Taken together, these findings indicate that JMJD8 functions as a positive regulator of TNF-induced NF-κB signaling.

No MeSH data available.


JMJD8 is required for IKK activation and RIP1 ubiquitination.(a) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 2.5, 5, 7.5, 10 and 15 minutes. Total cell lysates were prepared and immunoblotted with the indicated antibodies. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ, were normalized to IKK or α-Tubulin, and shown in relative to 0 minute of siControl (n = 2). (b) Control and JMJD8 knockdown HEK293T cells were induced with 1 μg/ml of GST-TNFα for 0, 5 and 10 minutes. TNFR1 complexes were pulled down with Glutathione beads and immunoblot for RIP1 and TNFα (n = 10). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Figs S9 and S10, respectively.
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f4: JMJD8 is required for IKK activation and RIP1 ubiquitination.(a) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 2.5, 5, 7.5, 10 and 15 minutes. Total cell lysates were prepared and immunoblotted with the indicated antibodies. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ, were normalized to IKK or α-Tubulin, and shown in relative to 0 minute of siControl (n = 2). (b) Control and JMJD8 knockdown HEK293T cells were induced with 1 μg/ml of GST-TNFα for 0, 5 and 10 minutes. TNFR1 complexes were pulled down with Glutathione beads and immunoblot for RIP1 and TNFα (n = 10). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Figs S9 and S10, respectively.

Mentions: Phosphorylation of IKK at Serine 177 and Serine 181 in the activation loop of IKKβ (Serine 176 and Serine 180 in IKKα) is required for its kinase activity23. To investigate the phosphorylation status of IKK, we therefore treated control and JMJD8-deficient cells with TNFα at indicated time points and measured the amount of p-IKK. Consistent with the IKK kinase assay, p-IKK was significantly lesser in JMJD8 knockdown cells (Fig. 4a).


JMJD8 is a positive regulator of TNF-induced NF- κ B signaling
JMJD8 is required for IKK activation and RIP1 ubiquitination.(a) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 2.5, 5, 7.5, 10 and 15 minutes. Total cell lysates were prepared and immunoblotted with the indicated antibodies. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ, were normalized to IKK or α-Tubulin, and shown in relative to 0 minute of siControl (n = 2). (b) Control and JMJD8 knockdown HEK293T cells were induced with 1 μg/ml of GST-TNFα for 0, 5 and 10 minutes. TNFR1 complexes were pulled down with Glutathione beads and immunoblot for RIP1 and TNFα (n = 10). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Figs S9 and S10, respectively.
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Related In: Results  -  Collection

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f4: JMJD8 is required for IKK activation and RIP1 ubiquitination.(a) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 2.5, 5, 7.5, 10 and 15 minutes. Total cell lysates were prepared and immunoblotted with the indicated antibodies. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ, were normalized to IKK or α-Tubulin, and shown in relative to 0 minute of siControl (n = 2). (b) Control and JMJD8 knockdown HEK293T cells were induced with 1 μg/ml of GST-TNFα for 0, 5 and 10 minutes. TNFR1 complexes were pulled down with Glutathione beads and immunoblot for RIP1 and TNFα (n = 10). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Figs S9 and S10, respectively.
Mentions: Phosphorylation of IKK at Serine 177 and Serine 181 in the activation loop of IKKβ (Serine 176 and Serine 180 in IKKα) is required for its kinase activity23. To investigate the phosphorylation status of IKK, we therefore treated control and JMJD8-deficient cells with TNFα at indicated time points and measured the amount of p-IKK. Consistent with the IKK kinase assay, p-IKK was significantly lesser in JMJD8 knockdown cells (Fig. 4a).

View Article: PubMed Central - PubMed

ABSTRACT

TNF-induced signaling mediates pleiotropic biological consequences including inflammation, immunity, cell proliferation and apoptosis. Misregulation of TNF signaling has been attributed as a major cause of chronic inflammatory diseases and cancer. Jumonji domain-containing protein 8 (JMJD8) belongs to the JmjC family. However, only part of the family members has been described as hydroxylase enzymes that function as histone demethylases. Here, we report that JMJD8 positively regulates TNF-induced NF-κB signaling. Silencing the expression of JMJD8 using RNA interference (RNAi) greatly suppresses TNF-induced expression of several NF-κB-dependent genes. Furthermore, knockdown of JMJD8 expression reduces RIP ubiquitination, IKK kinase activity, delays IκBα degradation and subsequently blocks nuclear translocation of p65. In addition, JMJD8 deficiency enhances TNF-induced apoptosis. Taken together, these findings indicate that JMJD8 functions as a positive regulator of TNF-induced NF-κB signaling.

No MeSH data available.