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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.


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JMJD8 positively regulates NF-κB.(a) HEK293T cells transfected with control and JMJD8 targeting siRNA oligos were treated with and without 10 ng/ml of TNFα for 0, 0.5, 2, 6 and 12 hours. The expression of TNFα, IL8, CCL5 and IκBα were measured by RT-qPCR (n = 4). (b) HEK293T cells transfected with control, JMJD8a and JMJD8b siRNA oligos were treated with and without 10 ng/ml of TNFα for 2 hours, the expression of TNFα and IL8 were measured by RT-qPCR. The knockdown expression of JMJD8 by each siRNA oligo was verified by immunobloting with a JMJD8 specific antibody (n = 4). (c) JMJD8 knockdown HEK293T cells reconstituted with JMJD8 were treated with TNFα for 2 hours and the expression of TNFα and IL8 were measured by RT-qPCR. The transient expression of ectopic JMJD8 was verified by immunobloting with a JMJD8 specific antibody (n = 4). (d) Control and JMJD8 knockdown HEK293T cells were infected with Sendai virus (150 HAU/ml) and the levels of IFNβ were measured by RT-qPCR (n = 4). Data represent the means ± SD. (*p > 0.05, **p > 0.01). Full-length blots are presented in Supplementary Fig. S3.
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f1: JMJD8 positively regulates NF-κB.(a) HEK293T cells transfected with control and JMJD8 targeting siRNA oligos were treated with and without 10 ng/ml of TNFα for 0, 0.5, 2, 6 and 12 hours. The expression of TNFα, IL8, CCL5 and IκBα were measured by RT-qPCR (n = 4). (b) HEK293T cells transfected with control, JMJD8a and JMJD8b siRNA oligos were treated with and without 10 ng/ml of TNFα for 2 hours, the expression of TNFα and IL8 were measured by RT-qPCR. The knockdown expression of JMJD8 by each siRNA oligo was verified by immunobloting with a JMJD8 specific antibody (n = 4). (c) JMJD8 knockdown HEK293T cells reconstituted with JMJD8 were treated with TNFα for 2 hours and the expression of TNFα and IL8 were measured by RT-qPCR. The transient expression of ectopic JMJD8 was verified by immunobloting with a JMJD8 specific antibody (n = 4). (d) Control and JMJD8 knockdown HEK293T cells were infected with Sendai virus (150 HAU/ml) and the levels of IFNβ were measured by RT-qPCR (n = 4). Data represent the means ± SD. (*p > 0.05, **p > 0.01). Full-length blots are presented in Supplementary Fig. S3.

Mentions: Our previous finding that methylation of p65 protein regulates its transcriptional activity10 prompted us to evaluate whether demethylases are also involved in TNF-induced NF-κB signaling. We did RNAi screening of a group of Jumonji domain-containing proteins and found that the JMJD8, a JmjC domain-only protein may be involved in regulating TNF-induced NF-κB signaling (Data not shown). To verify our observation, we compared the TNF-induced transcription kinetics of a few well-known NF-κB-dependent genes between control and JMJD8 knockdown HEK293T cells. As shown in Fig. 1a, the TNF-induced NF-κB transcriptional activity was almost completely abrogated in JMJD8 knockdown cells compared to the control cells. The effect of JMJD8 knockdown on TNF-induced NF-κB signaling was further supported by a NF-κB luciferase reporter assay (see Supplementary Fig. S1a).


JMJD8 is a positive regulator of TNF-induced NF- κ B signaling
JMJD8 positively regulates NF-κB.(a) HEK293T cells transfected with control and JMJD8 targeting siRNA oligos were treated with and without 10 ng/ml of TNFα for 0, 0.5, 2, 6 and 12 hours. The expression of TNFα, IL8, CCL5 and IκBα were measured by RT-qPCR (n = 4). (b) HEK293T cells transfected with control, JMJD8a and JMJD8b siRNA oligos were treated with and without 10 ng/ml of TNFα for 2 hours, the expression of TNFα and IL8 were measured by RT-qPCR. The knockdown expression of JMJD8 by each siRNA oligo was verified by immunobloting with a JMJD8 specific antibody (n = 4). (c) JMJD8 knockdown HEK293T cells reconstituted with JMJD8 were treated with TNFα for 2 hours and the expression of TNFα and IL8 were measured by RT-qPCR. The transient expression of ectopic JMJD8 was verified by immunobloting with a JMJD8 specific antibody (n = 4). (d) Control and JMJD8 knockdown HEK293T cells were infected with Sendai virus (150 HAU/ml) and the levels of IFNβ were measured by RT-qPCR (n = 4). Data represent the means ± SD. (*p > 0.05, **p > 0.01). Full-length blots are presented in Supplementary Fig. S3.
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f1: JMJD8 positively regulates NF-κB.(a) HEK293T cells transfected with control and JMJD8 targeting siRNA oligos were treated with and without 10 ng/ml of TNFα for 0, 0.5, 2, 6 and 12 hours. The expression of TNFα, IL8, CCL5 and IκBα were measured by RT-qPCR (n = 4). (b) HEK293T cells transfected with control, JMJD8a and JMJD8b siRNA oligos were treated with and without 10 ng/ml of TNFα for 2 hours, the expression of TNFα and IL8 were measured by RT-qPCR. The knockdown expression of JMJD8 by each siRNA oligo was verified by immunobloting with a JMJD8 specific antibody (n = 4). (c) JMJD8 knockdown HEK293T cells reconstituted with JMJD8 were treated with TNFα for 2 hours and the expression of TNFα and IL8 were measured by RT-qPCR. The transient expression of ectopic JMJD8 was verified by immunobloting with a JMJD8 specific antibody (n = 4). (d) Control and JMJD8 knockdown HEK293T cells were infected with Sendai virus (150 HAU/ml) and the levels of IFNβ were measured by RT-qPCR (n = 4). Data represent the means ± SD. (*p > 0.05, **p > 0.01). Full-length blots are presented in Supplementary Fig. S3.
Mentions: Our previous finding that methylation of p65 protein regulates its transcriptional activity10 prompted us to evaluate whether demethylases are also involved in TNF-induced NF-κB signaling. We did RNAi screening of a group of Jumonji domain-containing proteins and found that the JMJD8, a JmjC domain-only protein may be involved in regulating TNF-induced NF-κB signaling (Data not shown). To verify our observation, we compared the TNF-induced transcription kinetics of a few well-known NF-κB-dependent genes between control and JMJD8 knockdown HEK293T cells. As shown in Fig. 1a, the TNF-induced NF-κB transcriptional activity was almost completely abrogated in JMJD8 knockdown cells compared to the control cells. The effect of JMJD8 knockdown on TNF-induced NF-κB signaling was further supported by a NF-κB luciferase reporter assay (see Supplementary Fig. S1a).

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.


Related in: MedlinePlus