Role of IKKα in type I IFN-dependent and independent STAT1 phosphorylation induced by dsRNA.

<p>(A) HeLa cells were transfected with siRNA targeting IKKα or control siRNA for 48 h. Then, the cells were treated with an anti-IFNAR neutralizing antibody for 1 h followed by transfection with poly I:C (50 ng/well) for an additional 4 h. HeLa cells were also treated with IFN-β (200 IU/ml) for 15 min as a control to confirm the neutralizing activity of the anti-IFNAR antibody. (B) IFNAR-null U5A cells were transfected with siRNA targeting IKKα or control siRNA for 48 h, and the cells were then further transfected with poly I:C (500 ng/well) for 10 h. Cell extracts were analyzed by immunoblotting. The results are representative of three independent experiments.</p

Effects of IKKs on IFN-β expression in response to poly I:C.

<p>(A, C) HeLa cells were transfected with siRNA targeting IKKα, IKKβ, and IKKγ or control siRNA. Forty-eight hours after the transfection, the cells were further transfected with poly I:C (50 ng/well) for an additional 4 h. (A) The expression of IFN-β mRNA was analyzed by qRT-PCR. Data are presented as the mean ± SD of three independent experiments. *P<0.01. (B, D) Following IKKα RNAi silencing, the cells were transfected with poly I:C (50 ng/well) for 8 h (B) or up to 8 h (C). (B) IFN-β levels were determined by ELISA. All data are shown as the mean of three independent experiments. *P<0.01. (C, D) Protein levels were analyzed by immunoblotting. The results are representative of three independent experiments.</p

Non-Canonical Role of IKKα in the Regulation of STAT1 Phosphorylation in Antiviral Signaling

<div><p>Non-self RNA is recognized by retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), inducing type I interferons (IFNs). Type I IFN promotes the expression of IFN-stimulated genes (ISGs), which requires the activation of signal transducer and activator of transcription-1 (STAT1). We previously reported that dsRNA induced STAT1 phosphorylation via a type I IFN-independent pathway in addition to the well-known type I IFN-dependent pathway. IκB kinase α (IKKα) is involved in antiviral signaling induced by dsRNA; however, its role is incompletely understood. Here, we explored the function of IKKα in RLR-mediated STAT1 phosphorylation. Silencing of IKKα markedly decreased the level of IFN-β and STAT1 phosphorylation inHeH response to dsRNA. However, the inhibition of IKKα did not alter the RLR signaling-mediated dimerization of interferon responsive factor 3 (IRF3) or the nuclear translocation of nuclear factor-κB (NFκB). These results suggest a non-canonical role of IKKα in RLR signaling. Furthermore, phosphorylation of STAT1 was suppressed by IKKα knockdown in cells treated with a specific neutralizing antibody for the type I IFN receptor (IFNAR) and in IFNAR-deficient cells. Collectively, the dual regulation of STAT1 by IKKα in antiviral signaling suggests a role for IKKα in the fine-tuning of antiviral signaling in response to non-self RNA.</p></div

Effect of an IKK inhibitor on STAT1 phosphorylation in response to dsRNA.

<p>HeLa cells were pretreated with an IKK inhibitor at the indicated concentrations for 1 h and then transfected with poly I:C (50 ng/well) for 4 h. (A) The expression of IFN-β was analyzed by qRT-PCR. Data are presented as the mean ± SD of three independent experiments. *P<0.01. (B) Cell extracts were subjected to SDS-PAGE and then analyzed by immunoblotting. The results are representative of three independent experiments.</p

Influence of IKKα knockdown on JAK1 and Tyk2 activation in HeLa and U5A cells.

<p>Following knockdown of IKKα in HeLa (A) and U5A cells (B), poly I:C was introduced for an additional 4 h and 10 h, respectively. HeLa cells were treated with IFN-α (10 ng/ml) for 20 min to confirm phosphorylation of JAK1 and Tyk2 as a positive control. Cell extracts were analyzed by immunoblotting. The results are representative of three independent experiments.</p

No interaction of IKKα and STAT1.

<p>(A) HeLa cells were transfected with poly I:C for 4 h. (B) Twenty-four hours after transfection with a HA-tagged IKKα expression plasmid or an empty plasmid (mock), HeLa cells were transfected with poly I:C for 4 h. Immunoprecipitation (IP) was performed using an anti-STAT1, anti-IgG (A) or anti-HA beads (B), followed by immunoblotting using anti-IKKα, anti-STAT1 or anti-HA antibody. (C) The cells were fixed with 4% paraformaldehyde and incubated with anti-IKKα and anti-STAT1 antibodies. IKKα and STAT1 were then detected separately using a secondary antibody coupled to Alexa 488 (green) or Alexa 555 (red).</p

Comprehensive roles of IKKα in RLR signaling-mediated STAT1 activation.

<p>In addition to participating in the IKK complex to activate the canonical antiviral signaling pathway, IKKα mediates type I IFN expression (a long dashed dotted line: A). IKKα could directly activate STAT1 phosphorylation in a type I IFN-independent manner (a dashed line: B). IKKα-meditated cytokine expression likely activates STAT1 via a type I IFN-independent positive feedback loop (red line: C).</p

Effects of IKKα on translocation of the NFκB p65 subunit and dimerization of IRF3 in response to dsRNA.

<p>IKKα, IKKβ, or IKKγ in HeLa (A) and U5A (B) cells were silenced as described above. Then, the cells were transfected with poly I:C for 4 h. Cell extracts were subjected to SDS-PAGE or native-PAGE (IRF3) followed by immunoblotting. The results are representative of three independent experiments.</p

Effects of silencing IKKα on STAT1 phosphorylation in response to dsRNA transfection.

<p>(A) HeLa cells were transfected with poly I:C at the indicated concentrations for 4 h. (B) HeLa cells were transfected with poly I:C (50 ng/well) for up to 6 h or treated with TNFα for 10 min. (C) HeLa cells transiently overexpressing WT IKKα, IKKα S176A, or IKKα S180A were transfected with poly I:C for 4 h. Protein levels were analyzed by immunoblotting. The results are representative of three independent experiments.</p

Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

<div><p>Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-<i>O</i>-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. <i>In vitro</i> experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides <i>in vivo</i> in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.</p> </div