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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 deficiency reduces TNF-induced IκBα degradation and p65 translocation.(a) Control and JMJD8 knockdown HEK293T cells were treated with 10 ng/ml of TNFα for 0, 5, 15, 30, 45, 60 and 90 minutes. Cytoplasmic and nuclear fractions were prepared and immunoblotted for IκBα and p65. HSP90α and Lamin A/C were used as cytoplasmic and nuclear loading controls respectively. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ software, were normalized to HSP90α or Lamin A/C, and shown in relative to 0 minute of siControl (n = 3). (b) Control and JMJD8 knockdown HEK293T cells were treated with 10 ng/ml of TNFα for 30 minutes. P65 localization was visualized with an immunofluorescence assay. Images were acquired with an Olympus IX71 fluorescence microscope. Scale bar: 20 μm. (n = 3). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Fig. S4.
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f2: JMJD8 deficiency reduces TNF-induced IκBα degradation and p65 translocation.(a) Control and JMJD8 knockdown HEK293T cells were treated with 10 ng/ml of TNFα for 0, 5, 15, 30, 45, 60 and 90 minutes. Cytoplasmic and nuclear fractions were prepared and immunoblotted for IκBα and p65. HSP90α and Lamin A/C were used as cytoplasmic and nuclear loading controls respectively. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ software, were normalized to HSP90α or Lamin A/C, and shown in relative to 0 minute of siControl (n = 3). (b) Control and JMJD8 knockdown HEK293T cells were treated with 10 ng/ml of TNFα for 30 minutes. P65 localization was visualized with an immunofluorescence assay. Images were acquired with an Olympus IX71 fluorescence microscope. Scale bar: 20 μm. (n = 3). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Fig. S4.

Mentions: To dissect the role of JMJD8 in the TNF pathway, we first investigated the degradation of IκBα and the nuclear translocation of NF-κB, which are the two biochemical hallmarks of NF-κB activation. HEK293T cells were transfected with control or JMJD8-targeting siRNA oligos and treated with TNFα at the indicated time points. The cells were harvested and fractionated into cytoplasmic and nuclear fractions. TNF-induced degradation of IκBα peaked at 30 minutes followed by a resynthesis of IκBα at 90 minutes in the control cells (Fig. 2a upper panel). We found that IκBα degradation was reduced or delayed in the JMJD8-deficient cells and no resynthesis of IκBα was observed. Consistent with impaired IκBα degradation, we observed a significant reduction of TNF-induced p65 nuclear translocation in JMJD8-deficient cells (Fig. 2a, lower panel). To further confirm this observation, we performed an immunofluorescence assay to visualize the p65 subcellular localization in control and JMJD8 knockdown HEK293T cells with and without TNFα stimulation. Consistently, we noticed a complete blockage of p65 translocation into the nuclear of JMJD8 knockdown cells (Fig. 2b), indicating that JMJD8 is required for both IκBα degradation and the release of NF-κB into nucleus.


JMJD8 is a positive regulator of TNF-induced NF- κ B signaling
JMJD8 deficiency reduces TNF-induced IκBα degradation and p65 translocation.(a) Control and JMJD8 knockdown HEK293T cells were treated with 10 ng/ml of TNFα for 0, 5, 15, 30, 45, 60 and 90 minutes. Cytoplasmic and nuclear fractions were prepared and immunoblotted for IκBα and p65. HSP90α and Lamin A/C were used as cytoplasmic and nuclear loading controls respectively. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ software, were normalized to HSP90α or Lamin A/C, and shown in relative to 0 minute of siControl (n = 3). (b) Control and JMJD8 knockdown HEK293T cells were treated with 10 ng/ml of TNFα for 30 minutes. P65 localization was visualized with an immunofluorescence assay. Images were acquired with an Olympus IX71 fluorescence microscope. Scale bar: 20 μm. (n = 3). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Fig. S4.
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f2: JMJD8 deficiency reduces TNF-induced IκBα degradation and p65 translocation.(a) Control and JMJD8 knockdown HEK293T cells were treated with 10 ng/ml of TNFα for 0, 5, 15, 30, 45, 60 and 90 minutes. Cytoplasmic and nuclear fractions were prepared and immunoblotted for IκBα and p65. HSP90α and Lamin A/C were used as cytoplasmic and nuclear loading controls respectively. Relative intensity of bands were quantified using the Image Lab (BioRad)/ImageJ software, were normalized to HSP90α or Lamin A/C, and shown in relative to 0 minute of siControl (n = 3). (b) Control and JMJD8 knockdown HEK293T cells were treated with 10 ng/ml of TNFα for 30 minutes. P65 localization was visualized with an immunofluorescence assay. Images were acquired with an Olympus IX71 fluorescence microscope. Scale bar: 20 μm. (n = 3). Data represent means ± SD. (*p > 0.05). Full-length blots are presented in Supplementary Fig. S4.
Mentions: To dissect the role of JMJD8 in the TNF pathway, we first investigated the degradation of IκBα and the nuclear translocation of NF-κB, which are the two biochemical hallmarks of NF-κB activation. HEK293T cells were transfected with control or JMJD8-targeting siRNA oligos and treated with TNFα at the indicated time points. The cells were harvested and fractionated into cytoplasmic and nuclear fractions. TNF-induced degradation of IκBα peaked at 30 minutes followed by a resynthesis of IκBα at 90 minutes in the control cells (Fig. 2a upper panel). We found that IκBα degradation was reduced or delayed in the JMJD8-deficient cells and no resynthesis of IκBα was observed. Consistent with impaired IκBα degradation, we observed a significant reduction of TNF-induced p65 nuclear translocation in JMJD8-deficient cells (Fig. 2a, lower panel). To further confirm this observation, we performed an immunofluorescence assay to visualize the p65 subcellular localization in control and JMJD8 knockdown HEK293T cells with and without TNFα stimulation. Consistently, we noticed a complete blockage of p65 translocation into the nuclear of JMJD8 knockdown cells (Fig. 2b), indicating that JMJD8 is required for both IκBα degradation and the release of NF-κB into nucleus.

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.