DHX9 pairs with IPS-1 to sense double-stranded RNA in myeloid dendritic cells

The innate immune system is equipped with many molecular sensors for microbial DNA/RNA to quickly mount antimicrobial host immune responses. In this paper, we identified DHX9, a DExDc helicase family member, as an important viral dsRNA sensor in myeloid dendritic cells (mDCs). Knockdown of DHX9 expression by small heteroduplex RNA dramatically blocked the ability of mDCs to produce IFN-α/β and proinflammatory cytokines in response to polyinosine-polycytidylic acid, influenza A, and reovirus. DHX9 could specifically bind polyinosine-polycytidylic acid via its double-strand RNA binding motifs. DHX9 interacted with IPS-1 via the HelicC-HA2-DUF and CARD domains of DHX9 and IPS-1, respectively. Knockdown of DHX9 expression in mDCs blocked the activation of NF-κB and IFN regulatory factor 3 by dsRNA. Collectively, these results suggest that DHX9 is an important RNA sensor that is dependent on IPS-1 to sense pathogenic RNA.     

NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells

We previously demonstrated that indoxyl sulfate induces senescence and dysfunction of proximal tubular cells by activating p53 expression. However, little is known about the role of nuclear factor (NF)-κB in these processes. The present study examines whether activation (phosphorylation) of NF-κB by indoxyl sulfate promotes senescence and dysfunction in human proximal tubular cells (HK-2 cells). Indoxyl sulfate induced phosphorylation of NF-κB p65 on Ser-276, which was suppressed by N-acetylcysteine, an antioxidant. Furthermore, indoxyl sulfate induced NF-κB p65 expression. Inhibitors of NF-κB (pyrrolidine dithiocarbamate and isohelenin) and NF-κB p65 small interfering RNA (siRNA) suppressed indoxyl sulfate-induced senescence-associated β-galactosidase activity and expression of p53, transforming growth factor (TGF)-β1, and α-smoothe muscle actin (SMA). The induction of p53 expression and p53 promoter activity by indoxyl sulfate were inhibited by pifithrin-α, p-nitro, an inhibitor of p53, whereas p53-transfected cells showed enhanced p53 promoter activity. NF-κB inhibitors suppressed indoxyl sulfate-induced p21 expression, whereas NF-κB p65 siRNA enhanced its expression. NF-κB inhibitors partially alleviated indoxyl sulfate-induced inhibition of cellular proliferation. NF-κB p65 siRNA-transfected cells showed less proliferation in the presence of indoxyl sulfate than control cells. Phosphorylated NF-κB p65 was expressed and colocalized with p53, p21, β-galactosidase, TGF-β1, and α-SMA in the kidneys of chronic renal failure (CRF) rats. AST-120, which reduces serum indoxyl sulfate level, suppressed their expression in the CRF rat kidneys. Taken together, NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells. More notably, indoxyl sulfate accelerates proximal tubular cell senescence with progression of CRF through reactive oxygen species-NF-κB-p53 pathway.     

Human β-defensin-3 and nuclear factor-kappa B p65 synergistically promote the cell proliferation and invasion of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a usual oral cancer. Therefore, it's essential to identify targets for its early diagnosis and therapy. This research aimed to explore the roles of human β-defensin-3 (hBD-3) and nuclear factor-kappa B (NF-κB) p65 in the pathogenesis and progression of OSCC. The connection between NF-κB p65 and the carcinogenesis of oral cancer was analyzed by immunohistochemical staining. The relative expressions of hBD-3 and NF-κB p65 in OSCC cells were evaluated by qRT-PCR and Western blot. Afterward, hBD-3 was knocked down, and NF-κB p65 was overexpressed. The cell viability and invasion were tested via CCK-8 and Transwell experiment, and the expression of hBD-3, NF-κB p65, and its downstream molecules was evaluated by Western blot. The expression of NF-κB p65 was increased with the aggravation of the oral submucosal fibrosis. HBD-3 and NF-κB p65 were high-expressed in OSCC cells. The viability and invasion abilities of OSCC cells that knocked down hBD-3 were markedly decreased, while they were restored by the overexpression of NF-κB p65. The expressions of NF-κB p65 and c-myc were diminished while IκB and p21 were raised with the knockdown of hBD-3. After overexpression of NF-κB p65, the expression of hBD-3 and IκB did not change markedly, while c-myc was increased and p21 was decreased dramatically. HBD-3 and NF-κB p65 facilitate the proliferation and invasion of OSCC cells, and hBD-3 may promote this process by governing the expression of NF-κB p65 and its downstream c-myc and p21.     

LncRNA C9orf139 can regulate the progression of esophageal squamous carcinoma by mediating the miR-661/HDAC11 axis

Increasing evidence has indicated that long non-coding RNAs (LncRNAs) play multiple functions in the development of cancer and function as indicators of diagnosis and prognosis. This aim of this study was to investigate the roles LncRNA C9orF139 had in the progression of esophageal squamous carcinoma (ESCC). We found C9orf139 was highly expressed in ESCC and knock down the expression of C9orf139 significantly suppressed cell proliferation, promoted apoptosis, and inhibited migration and invasion. C9orf139 was able to negatively regulate miR-661 expression. At the same time, HDAC11 expression was negatively regulated by miR-661. The C9orf139/miR-661/HDAC11 axis was further involved in regulating the expression of the NF-κB signaling pathway. The association between the C9orf139 knockdown and the reduced tumor growth and size was observed during in vivo study. C9orf139 is highly expressed in ESCC, and is thus qualified to be used as a potential diagnostic and prognostic marker for ESCC. Its promotion of ESCC progression is achieved by mediating the miR-661/HDAC11 axis.     

Diacylglycerol kinase α enhances protein kinase Cζ-dependent phosphorylation at Ser311 of p65/RelA subunit of nuclear factor-κB

We recently reported that diacylglycerol kinase (DGK) α enhanced tumor necrosis factor-α (TNF-α)-induced activation of nuclear factor-κB (NF-κB). However, the signaling pathway between DGKα and NF-κB remains unclear. Here, we found that small interfering RNA-mediated knockdown of DGKα strongly attenuated protein kinase C (PKC) ζ-dependent phosphorylation of a large subunit of NF-κB, p65/RelA, at Ser311 but not PKCζ-independent phosphorylation at Ser468 or Ser536. Moreover, knockdown and overexpression of PKCζ suppressed and synergistically enhanced DGKα-mediated NF-κB activation, respectively. These results strongly suggest that DGKα positively regulates TNF-α-dependent NF-κB activation via the PKCζ-mediated Ser311 phosphorylation of p65/RelA.     

NOS2 regulation of LPS-induced airway inflammation via S-nitrosylation of NF-{kappa}B p65

Inducible nitric oxide synthase (NOS2) expression is increased in the airway epithelium in acute inflammatory disorders although the physiological impact remains unclear. We have previously shown that NOS2 inhibits NF-κB (p50-p65) activation in respiratory epithelial cells by inducing S-nitrosylation of the p65 monomer (SNO-p65). In addition, we have demonstrated that mouse lung SNO-p65 levels are acutely depleted in a lipopolysaccharide (LPS) model of lung injury and that augmenting SNO-p65 levels before LPS treatment results in decreased airway epithelial NF-κB activation, airway inflammation, and lung injury. We now show that aerosolized LPS induces NOS2 expression in the respiratory epithelium concomitant with an increase in lung SNO-p65 levels and a decrease in airway NF-κB activity. Genetic deletion of NOS2 results in an absence of SNO-p65 formation, persistent NF-κB activity in the respiratory epithelium, and prolonged airway inflammation. These results indicate that a primary function of LPS-induced NOS2 expression in the respiratory epithelium is to modulate the inflammatory response through deactivation of NF-κB via S-nitrosylation of p65, thereby counteracting the initial stimulus-coupled denitrosylation.     

Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment

The DNA binding activity of NF-κB is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-κB activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF. The phosphorylation and subsequent degradation of I-κBα as well as the serine 536 phosphorylation and nuclear translocation of p65 NF-κB were insufficient for VCAM-1 expression in response to TNF. The requirement for p50 NF-κB in TNF-induced VCAM-1 expression may be associated with its interaction with and phosphorylation by DNA-PK, which appears to be dominant over the requirement for p65 NF-κB activation. p50 NF-κB binding to its consensus sequence increased its susceptibility to phosphorylation by DNA-PK. Additionally, DNA-PK activity appeared to increase the association between p50/p50 and p50/p65 NF-κB dimers upon binding to DNA and after binding of p50 NF-κB to the VCAM-1 promoter. Analyses of the p50 NF-κB protein sequence revealed that both serine 20 and serine 227 at the amino terminus of the protein are putative sites for phosphorylation by DNA-PK. Mutation of serine 20 completely eliminated phosphorylation of p50 NF-κB by DNA-PK, suggesting that serine 20 is the only site in p50 NF-κB for phosphorylation by DNA-PK. Re-establishing wild-type p50 NF-κB, but not its serine 20/alanine mutant, in p50 NF-κB(-/-) fibroblasts reversed VCAM-1 expression after TNF treatment, demonstrating the importance of the serine 20 phosphorylation site in the induction of VCAM-1 expression. Together, these results elucidate a novel mechanism for the involvement of DNA-PK in the positive regulation of p50 NF-κB to drive VCAM-1 expression.     

Curcumin Attenuates gp120-Induced Microglial Inflammation by Inhibiting Autophagy via the PI3K Pathway

Microglial inflammation plays an essential role in the pathogenesis of HIV-associated neurocognitive disorders. A previous study indicated that curcumin relieved microglial inflammatory responses. However, the mechanism of this process remained unclear. Autophagy is a lysosome-mediated cell content-dependent degradation pathway, and uncontrolled autophagy leads to enhanced inflammation. The role of autophagy in curcumin-attenuating BV2 cell inflammation caused by gp120 was investigated with or without pretreatment with the autophagy inhibitor 3-MA and blockers of NF-κB, IKK, AKT, and PI3K, and we then detected the production of the inflammatory mediators monocyte chemoattractant protein-1 (MCP-1) and IL17 using ELISA, and autophagy markers ATG5 and LC3 II by Western Blot. The autophagic flux was observed by transuding mRFP-GFP-LC3 adenovirus. The effect of the blockers on gp120-induced BV2 cells was examined by the expression of p-AKT, p-IKK, NF-κB, and p65 in the nuclei and LC3 II and ATG5. gp120 promoted the expression of MCP-1 and IL-17, enhanced autophagic flux, and up-regulated the expression of LC3 II and ATG5, while the autophagy inhibitor 3-MA down-regulated the phenomena above. Curcumin has similar effects with 3-MA, in which curcumin inhibited NF-κB by preventing the translocation of NF-κB p65. Curcumin also inhibited the phosphorylation of p-PI3K, p-AKT, and p-IKK, which leads to down-regulation of NF-κB. Curcumin reduced autophagy via PI3K/AKT/IKK/NF-κB, thereby reducing BV2 cellular inflammation induced by gp120.