Dynein Light Chain LC8 Alleviates Nonalcoholic Steatohepatitis By Regulating Nuclear Factor κB Through Interference With IκBα Phosphorylation

Nonalcoholic steatohepatitis (NASH) is a liver disease characterized by fat accumulation and chronic inflammation in the liver. Although dynein light chain of 8 kDa (LC8) was identified previously as an inhibitor of nuclear factor kappa B (NF-κB), a key regulator of inflammation, its role in NASH remains unknown. In this study, we investigated whether LC8 can alleviate NASH using a mouse model of methionine and choline-deficient (MCD) diet–induced NASH and examined the underlying mechanism. LC8 transgenic (Tg) mice showed lower hepatic steatosis and less progression of NASH, including inflammation, oxidative stress, and hepatic fibrosis, compared to wild-type (WT) mice after consuming an MCD diet. The hepatic expression of lipogenic genes was lower, while that of lipolytic and mitochondrial genes was greater in LC8 Tg mice than WT mice, which might be associated with resistance of LC8 Tg mice to hepatic steatosis. Consumption of an MCD diet enhanced IκBα phosphorylation and subsequent p65 liberation from IκBα and nuclear translocation, resulting in induction of NF-κB targets, including pro-inflammatory cytokines and chemokines. However, these effects of MCD diet were reduced by LC8 overexpression. Taken together, these results suggest that LC8 alleviates MCD diet–induced NASH by inhibiting NF-κB through binding to IκBα to interfere with IκBα phosphorylation.

PIM2 promotes lung adenocarcinoma cell migration by regulating XIAP/NF-κB pathway

Background and objective: Proviral insertion site in Moloney murine leukemia virus (PIM)2 functions as a serine/threonine kinase to participate in regulating cell proliferation and cell cycle. PIM2 has been shown to be elevated in the lung cancer cell lines. This study was performed to investigate the role of PIM2 in lung adenocarcinoma cell growth. Mateial and methods: Expression level of PIM2 in lung adenocarcinoma tissues and cells was detected by qRT-PCR (quantitative Reverse Transcription PCR) and western blot. The over-expression and knockdown of PIM2 were separately established by employing pcDNA and siRNA to explore the effects on the cell viability, apoptosis, invasion and migration. The downstream pathways were evaluated by western blot assay. Results: Lung adenocarcinoma tissues and cells showed an elevation of both PIM2 mRNA and protein expression. Knocking down PIM2 decreased the cell viability and promoted the apoptosis, which can be reversed by pcDNA-mediated over-expression of PIM2. PIM2 silencing suppressed the promotional effect of over-expression of PIM2 on cell invasion and migration through increasing IκBα expression and decreasing the X-linked inhibitor of apoptosis protein (XIAP), p65 and IκBα phosphorylation. While, over-expression of PIM2 showed opposite effect on IκBα and XIAP expression or p65 and IκBα phosphorylation. Conclusion: PIM2 can not only suppress lung adenocarcinoma cell apoptosis but also promote cell migration and invasion depending on XIAP/NF-κB signaling pathway.

Methyl jasmonate reduces the inflammation and apoptosis of HK-2 cells induced by LPS by regulating the NF-κB pathway

Background: Methyl jasmonate is a bioactive oxylipid that participates in the defense-related mechanisms of plants. The anti-inflammatory and anti-oxidative capacities of methyl jasmonate against lipopolysaccharide (LPS) induced arthritis have been widely investigated. However, the role of methyl jasmonate in LPS-induced cell model of tubular-interstitial nephritis (TIN) has not been reported. Methods: LPS (5 µg/mL) was applied to treat human renal tubular epithelial cell line (HK-2) for the establishment of TIN cell model. LPS-induced HK-2 was incubated with 10 or 20 µM methyl jasmonate, cell viability and apoptosis were assessed by MTT and flow cytometry. ELISA and qRT-PCR were performed to determine the levels of interleukin (IL)-1 beta (IL-1β), IL-6, IL-8 and tumor necrosis factor-α (TNF-α). The downstream pathway was investigated by western blot. Results: LPS induced cytotoxicity in HK-2 cell accompanied by decrease of cell viability and increase of cell apoptosis. Methyl jasmonate dosage dependently enhanced the cell viability and reduced cell apoptosis to ameliorate the cytotoxicity. LPS also induced inflammatory response in HK-2 cell with increased IL-1β, IL-6, IL-8 and TNF-α. Methyl jasmonate attenuated LPS-induced inflammation in HK-2 cell. Protein expression of IκBα was down-regulated, p65 and IκBα phosphorylation were up-regulated in LPS-induced HK-2. Methyl jasmonate attenuated LPS-induced decrease of IκBα and increase of p65 and IκBα phosphorylation in HK-2 cell. Conclusion: Methyl jasmonate demonstrated anti-apoptotic and anti-inflammatory effects on LPS-induced HK-2 cell through suppression of NF-κB activation.

Methyl jasmonate reduces the inflammation and apoptosis of HK-2 cells induced by LPS by regulating the NF-κB pathway

Background: Methyl jasmonate is a bioactive oxylipid that participates in the defense-related mechanisms of plants. The anti-inflammatory and anti-oxidative capacities of methyl jasmonate against lipopolysaccharide (LPS) induced arthritis have been widely investigated. However, the role of methyl jasmonate in LPS-induced cell model of tubular-interstitial nephritis (TIN) has not been reported. Methods: LPS (5 µg/mL) was applied to treat human renal tubular epithelial cell line (HK-2) for the establishment of TIN cell model. LPS-induced HK-2 was incubated with 10 or 20 µM methyl jasmonate, cell viability and apoptosis were assessed by MTT and flow cytometry. ELISA and qRT-PCR were performed to determine the levels of interleukin (IL)-1 beta (IL-1β), IL-6, IL-8 and tumor necrosis factor-α (TNF-α). The downstream pathway was investigated by western blot. Results: LPS induced cytotoxicity in HK-2 cell accompanied by decrease of cell viability and increase of cell apoptosis. Methyl jasmonate dosage dependently enhanced the cell viability and reduced cell apoptosis to ameliorate the cytotoxicity. LPS also induced inflammatory response in HK-2 cell with increased IL-1β, IL-6, IL-8 and TNF-α. Methyl jasmonate attenuated LPS-induced inflammation in HK-2 cell. Protein expression of IκBα was down-regulated, p65 and IκBα phosphorylation were up-regulated in LPS-induced HK-2. Methyl jasmonate attenuated LPS-induced decrease of IκBα and increase of p65 and IκBα phosphorylation in HK-2 cell. Conclusion: Methyl jasmonate demonstrated anti-apoptotic and anti-inflammatory effects on LPS-induced HK-2 cell through suppression of NF-κB activation.

Novel Histone Deacetylase 6 Inhibitor CKD-506 Inhibits NF-κB Signaling in Intestinal Epithelial Cells and Macrophages and Ameliorates Acute and Chronic Murine Colitis

Background Selective blocking of HDAC6 has become a promising strategy in treating inflammatory bowel disease. CKD-506 is a novel isoform-selective inhibitor of histone deacetylase 6. The present study was performed to evaluate the effect of CKD-506 on the NF-κB signaling pathway in intestinal epithelial cells (IECs) and macrophages and on murine models of acute and chronic colitis. Methods RAW264RAW264.7 murine macrophages and COLO 205 human IECs were pretreated with CKD-506 and then stimulated with lipopolysaccharides (LPS). Cytokine expression of TNF-α, interleukin (IL)-6, IL-8, and IL-10 was measured by ELISA. The effect of CKD-506 on NF-κB signaling was evaluated by Western blotting of IκBα phosphorylation/degradation and electrophoretic mobility shift assay. In vivo studies were performed using a dextran sulfate sodium (DSS)–induced acute colitis model, a chronic colitis model in IL-10 knockout mice, and an adoptive transfer model. Colitis was quantified by the disease activity index, colon length, and histopathologic evaluation. Results CKD-506 suppressed the expression of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α in IECs and macrophages. CKD-506 strongly inhibited IκBα phosphorylation/degradation and the DNA-binding activity of NF-κB. Oral administration of CKD-506 attenuated DSS-induced acute colitis and chronic colitis in IL-10-/- and adoptive transfer models. CKD-506 ameliorated weight loss, disease activity, and histopathologic score in colitis mice and downregulated IκBα phosphorylation and pro-inflammatory cytokine production significantly. Conclusions CKD-506 blocked NF-κB signaling in IECs and macrophages and ameliorated experimental acute and chronic murine colitis models, which suggests that CKD-506 is a promising candidate for inflammatory bowel disease treatment as a small molecular medicine.

NF-κB activation is a turn on for vaccinia virus phosphoprotein A49 to turn off NF-κB activation

Vaccinia virus protein A49 inhibits NF-κB activation by molecular mimicry and has a motif near the N terminus that is conserved in IκBα, β-catenin, HIV Vpu, and some other proteins. This motif contains two serines, and for IκBα and β-catenin, phosphorylation of these serines enables recognition by the E3 ubiquitin ligase β-TrCP. Binding of IκBα and β-catenin by β-TrCP causes their ubiquitylation and thereafter proteasome-mediated degradation. In contrast, HIV Vpu and VACV A49 are not degraded. This paper shows that A49 is phosphorylated at serine 7 but not serine 12 and that this is necessary and sufficient for binding β-TrCP and antagonism of NF-κB. Phosphorylation of A49 S7 occurs when NF-κB signaling is activated by addition of IL-1β or overexpression of TRAF6 or IKKβ, the kinase needed for IκBα phosphorylation. Thus, A49 shows beautiful biological regulation, for it becomes an NF-κB antagonist upon activation of NF-κB signaling. The virulence of viruses expressing mutant A49 proteins or lacking A49 (vΔA49) was tested. vΔA49 was attenuated compared with WT, but viruses expressing A49 that cannot bind β-TrCP or bind β-TrCP constitutively had intermediate virulence. So A49 promotes virulence by inhibiting NF-κB activation and by another mechanism independent of S7 phosphorylation and NF-κB antagonism. Last, a virus lacking A49 was more immunogenic than the WT virus.