A20 attenuates hypoxia-induced pulmonary arterial hypertension by inhibiting NF-κB activation and pulmonary artery smooth muscle cell proliferation.

PAH is a progressive disease characterized by uncontrolled proliferation of PASMCs. Zinc finger protein A20 is a negative feedback regulatory protein of NF-κB activity. The aim of this study was to evaluate zinc finger protein A20 can alleviate PAH in hypoxia exposed mice. C57BL/6 mice received a tail vein injection of adenovirus-mediated ad-A20 and ad-A20 shRNA were exposed to hypoxia. PASMCs isolated from rat pulmonary arteries were cultured in hypoxia, and were transfection of A20 adenovirus. Pulmonary hemodynamic parameters were measured by right heart catheterization. Pulmonary vascular morphological changes were analyzed by HE and α-SMA staining. The expression changes of A20, NF-κB and its downstream protein were detected. The expression of phospho-p65 was increased with the prolongation of hypoxia time. The expression of A20 in lung tissue of chronic hypoxia group decreased with the prolongation of hypoxia time. Adenovirus-mediated A20 (ad-A20) overexpression significantly attenuated the abnormally increased RVSP, RV/(LV + S) ratio, WT%, WA%, α-SMA and the pulmonary vessel muscularization. Ad-A20 treatment markedly attenuated the degradation of phospho-p65 and inhibited the induction of phospho-IκBα induced by hypoxia treatment. Furthermore, silencing A20 abolished the protection by anti-inflammatory activity and the inhibitory effect on cell proliferation. We showed that Zinc finger protein A20 can block NF-κB signaling pathway, alleviates the hypoxia-induced abnormal elevation of pulmonary arterial pressure, hyperproliferation of PASMCs and the pulmonary vascular remodeling.

Downregulation of A20 increases the cytotoxicity of IFN-γ in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a highly fatal disease mandating development of novel, effective therapeutic strategy. Interferon-gamma (IFN-γ) is a pleiotropic cytokine with immunomodulatory, antiviral, and antitumor effects. Although IFN-γ is a promising antitumor agent, its application is limited by resistance in tumor cells. A20 is a zinc-finger protein that was initially identified as a gene product induced by tumor necrosis factor α in human umbilical vein endothelial cells. In this study, we found that silencing of A20 combined with IFN-γ significantly represses cell viability, and induces apoptosis and cell-cycle arrest in HCC cells. By investigating mechanisms implicated in A20 and IFN-γ-mediated signaling pathways, we revealed that the phosphoinositide 3-kinase/Akt signaling pathway and antiapoptotic B-cell lymphoma 2 proteins were repressed. Moreover, we also found that phosphorylation of STAT1 and STAT3 was significantly enhanced after the downregulation of A20 in combination with treatment of IFN-γ. Inhibitor of STAT1 but not STAT3 could block the antitumor effect of IFN-γ. Therefore, targeting A20 enhances the cytotoxicity of IFN-γ against HCC cells and may present a promising therapeutic strategy for HCC.

A20, an essential component of the ubiquitin-editing protein complex, is a negative regulator of inflammation in human myometrium and foetal membranes.

STUDY QUESTION: Does A20 regulate mediators involved in the terminal processes of human labour in primary myometrial and amnion cells? SUMMARY ANSWER: A20 is a nuclear factor-kappa B (NF-κB) responsive gene that acts as a negative regulator of NF-κB-induced expression of pro-labour mediators. WHAT IS KNOWN ALREADY: Inflammation is commonly implicated in spontaneous preterm birth and the processes involved in rupture of foetal membranes and uterine contractions. In myometrium and foetal membranes, the pro-inflammatory transcription factor NF-κB regulates the transcription of pro-labour mediators in response to inflammatory stimuli. In non-gestational tissues, A20 is widely recognised as an anti-inflammatory protein that inhibits inflammation-induced NF-κB signalling. STUDY DESIGN, SIZE, DURATION: Primary human amnion and myometrial cells were used to determine the effect of pro-inflammatory mediators on A20 expression and the effect of A20 siRNA on the expression and secretion of pro-labour mediators. The expression of A20 was assessed in myometrium and foetal membranes from non-labouring and labouring women at preterm and or term (n = 8 or nine samples per group). PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects of pro-inflammatory mediators and of A20 siRNA in cell cultures were determined by quantitative RT-PCR (qRT-PCR), western blots, immunoassays, gelatin zymography and luciferase assays. A20 expression in tissue samples was assessed by qRT-PCR. Statistical significance was ascribed to a P value < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: In primary cells isolated from myometrium and or amnion, the pro-inflammatory cytokines IL1B and TNF, the bacterial products flagellin and fsl-1, and the viral double stranded RNA analogue poly(I:C) significantly increased A20 mRNA expression via NF-κB. A20 siRNA studies in primary myometrial and amnion cells demonstrated an augmentation of inflammation-induced expression and or secretion of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL1, CXCL8, CCL2), adhesion molecules (ICAM1, VCAM1), contraction-associated proteins (PTGS2, PTGFR, PGF2α) and the extracellular matrix degrading enzyme MMP9, as well as NF-κB activation. Inhibition of NF-κB activity significant attenuated inflammation-induced expression of pro-labour mediators in A20 siRNA transfected cells. Finally, A20 mRNA expression was decreased in myometrium and foetal membranes with labour, and in foetal membranes with chorioamnionitis. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The conclusions of this study are solely reliant on the data from in vitro experiments using cells isolated from myometrium and amnion. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study raise the possibility that targeting A20 may be a therapeutic approach to reduce inflammation associated with spontaneous preterm birth. STUDY FUNDING AND COMPETING INTEREST(S): Associate Professor Martha Lappas is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; grant no. 1047025). Funding for this study was provided by the NHMRC (grant no. 1058786), Norman Beischer Medical Research Foundation and the Mercy Research Foundation. There are no competing interests.

MiR-125b regulates proliferation and apoptosis of nasopharyngeal carcinoma by targeting A20/NF-κB signaling pathway.

MiR-125b is aberrantly expressed and has a role in the various types of tumors. However, the role and mechanism of miR-125b in nasopharyngeal carcinoma (NPC) are unclear. In this study, we investigated the role and mechanism of miR-125b in NPC. We observed that miR-125b was significantly upregulated in the NPC tissues relative to normal nasopharyngeal mucosa (NNM), and its increment was correlated with poor patient survival, and was an independent predictor for reduced patient survival; miR-125b promoted NPC cell proliferation and inhibited NPC cell apoptosis; in a mouse model, administration of miR-125b antagomir significantly reduced the growth of NPC xenograft tumors. Mechanistically, we confirmed that A20 was a direct target of miR-125b, and found that activation of nuclear factor κB (NF-κB) signaling pathway by A20 mediated miR-125b-promoting NPC cell proliferation and -inhibiting NPC cell apoptosis. With a combination of loss-of-function and gain-of-function approaches, we further showed that A20 inhibited NPC cell proliferation, induced NPC cell apoptosis, and reduced the growth of NPC xenograft tumors. Moreover, A20 was significantly downregulated, whereas p-p65(RelA) was significantly upregulated in the NPC tissues relative to normal nasopharyngeal mucosa, and miR-125b level was negatively associated with A20 level, whereas positively associated with p-p65 level. Our data demonstrate that miR-125b regulates NPC cell proliferation and apoptosis by targeting A20/NF-κB signaling pathway, and miR-125b acts as oncogene, whereas A20 functions as tumor suppressor in NPC, highlighting the therapeutic potential of miR-125b/A20/NF-κB signaling axis in the NPC.

Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1ß instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation.

Zinc finger protein A20 is involved in the antipsoriatic effect of calcipotriol.

BACKGROUND: Calcipotriol ameliorates psoriasis through inducing keratinocyte apoptosis and inhibiting nuclear factor kappa B (NF-κB) activation, while zinc finger protein A20 exhibits an anti-apoptotic effect on various types of cells. OBJECTIVES: To understand the potential role of A20 in calcipotriol function. MATERIALS AND METHODS: The A20 levels were evaluated in the psoriatic skins from both human patients and K14-vascular endothelial growth factor (VEGF) transgenic mice that received or did not receive topical calcipotriol treatment. The in vitro effect of calcipotriol on A20 expression and the downstream NF-κB pathway was studied using a model of human foreskin keratinocytes (HFKs) that were stimulated with psoriatic cytokines [M5, a cocktail of interleukin (IL)-1a, IL-17A, IL-22, Oncostatin M and tumour necrosis factor-α, each at 10 ng mL(-1) ]. RESULTS: A20 expression was enhanced in both psoriatic tissues and keratinocytes when compared with controls, but decreased on calcipotriol treatment. The transfection of A20 small interfering RNA (siRNA) improved cell differentiation, and inhibited psoriatic inflammation in a HFK model. Moreover, the nuclear expression of NF-κB p65 decreased on A20 downregulation in psoriatic tissues and keratinocytes. Interestingly, calcipotriol enhanced the binding of A20 to ring finger protein 114 (RNF114) and A20-binding inhibitor of NF-κB-1 (ABIN-1) in HFKs, two negative regulators of the NF-κB pathway. CONCLUSIONS: Calcipotriol exhibits its antipsoriatic function through suppressing A20 expression and stabilizing negative regulators of the NF-κB pathway.