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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 TNF-induced IKK kinase activity.(a) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 5, 10, 15 and 30 minutes. IKK kinase activity was measured with an in vitro kinase assay followed by immunoblotting using the anti-p-IκBα and anti-IKKβ antibodies. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ, were normalized to IKKβ, and shown in relative to 0 minute of siControl (n = 2). (b) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 15, 30, 45 and 90 minutes. Total cell lysates were prepared and immunoblotted with the indicated antibodies (n = 2). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Figs S5–S8, respectively.
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f3: JMJD8 is required for TNF-induced IKK kinase activity.(a) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 5, 10, 15 and 30 minutes. IKK kinase activity was measured with an in vitro kinase assay followed by immunoblotting using the anti-p-IκBα and anti-IKKβ antibodies. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ, were normalized to IKKβ, and shown in relative to 0 minute of siControl (n = 2). (b) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 15, 30, 45 and 90 minutes. Total cell lysates were prepared and immunoblotted with the indicated antibodies (n = 2). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Figs S5–S8, respectively.

Mentions: The observed defect in IκBα degradation in JMJD8 knockdown cells suggests that JMJD8 may regulate the upstream signal transduction of TNF pathway. IκBα phosphorylation by IKK complexes is a prerequisite step for IκBα degradation7. Therefore, we investigated the activation of IKK in the presence or absence of JMJD8. We treated the control and JMJD8 knockdown HEK293T cells with TNFα at indicated time points and measured the IKK kinase activity with an in vitro IKK kinase assay. IKK kinase activity was detected as early as 5 minutes post-TNF stimulation and peaked at 10 minutes (Fig. 3a, lower panel). In the absence of JMJD8, TNF-induced IKK activation was significantly reduced as measured by the in vitro IKK kinase assay as well as the immunoblotting of p-IκBα in the total cell extracts (Fig. 3a, upper panel). This result suggests that JMJD8 is required for TNF-induced activation of IKK.


JMJD8 is a positive regulator of TNF-induced NF- κ B signaling
JMJD8 is required for TNF-induced IKK kinase activity.(a) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 5, 10, 15 and 30 minutes. IKK kinase activity was measured with an in vitro kinase assay followed by immunoblotting using the anti-p-IκBα and anti-IKKβ antibodies. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ, were normalized to IKKβ, and shown in relative to 0 minute of siControl (n = 2). (b) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 15, 30, 45 and 90 minutes. Total cell lysates were prepared and immunoblotted with the indicated antibodies (n = 2). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Figs S5–S8, respectively.
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f3: JMJD8 is required for TNF-induced IKK kinase activity.(a) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 5, 10, 15 and 30 minutes. IKK kinase activity was measured with an in vitro kinase assay followed by immunoblotting using the anti-p-IκBα and anti-IKKβ antibodies. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ, were normalized to IKKβ, and shown in relative to 0 minute of siControl (n = 2). (b) Control and JMJD8 knockdown HEK293T cells were induced with 10 ng/ml of TNFα for 0, 15, 30, 45 and 90 minutes. Total cell lysates were prepared and immunoblotted with the indicated antibodies (n = 2). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Figs S5–S8, respectively.
Mentions: The observed defect in IκBα degradation in JMJD8 knockdown cells suggests that JMJD8 may regulate the upstream signal transduction of TNF pathway. IκBα phosphorylation by IKK complexes is a prerequisite step for IκBα degradation7. Therefore, we investigated the activation of IKK in the presence or absence of JMJD8. We treated the control and JMJD8 knockdown HEK293T cells with TNFα at indicated time points and measured the IKK kinase activity with an in vitro IKK kinase assay. IKK kinase activity was detected as early as 5 minutes post-TNF stimulation and peaked at 10 minutes (Fig. 3a, lower panel). In the absence of JMJD8, TNF-induced IKK activation was significantly reduced as measured by the in vitro IKK kinase assay as well as the immunoblotting of p-IκBα in the total cell extracts (Fig. 3a, upper panel). This result suggests that JMJD8 is required for TNF-induced activation of IKK.

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.