NDR1/STK38 potentiates NF‐κB activation by its kinase activity

Human NDR1/STK38 belongs to the nuclear‐Dbf2‐related (NDR) family of Ser/Thr kinases. It has been implicated to function in centrosome duplication, control of cell cycle and apoptosis. However, the mechanism of NDR1 signaling pathway remains largely elusive. Here, we report a novel role of NDR1 in NF‐κB activation. By overexpression, NDR1 potentiates NF‐κB activation induced by TNFα, whereas knockdown of NDR1 expression inhibits NF‐κB activation induced by TNFα. Coimmunoprecipitation shows that NDR1 interacts with multiple signal components except p65 in NF‐κB signaling pathway. Furthermore, both phosphorylation and kinase dead mutants of NDR1 lose their synergistic effects on TNFα‐induced NF‐κB activation. siRNA oligo against NDR1 and kinase dead mutant as well mainly block the NF‐κB activation induced by TRAF2 but not RIP1. Furthermore, kinase dead mutant of NDR1 fails to interact with TRAF2. Taken together, our findings suggest an unknown function of NDR1, which may regulate NF‐κB activation by its kinase activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Pinitol attenuates LPS‐induced pneumonia in experimental animals: Possible role via inhibition of the TLR‐4 and NF‐κB/IκBα signaling cascade pathway

Pneumonia is a chronic disorder of the respiratory system associated with worsening quality of life and a significant economic burden. Pinitol, a plant cyclic polyol, has been documented for immune‐inflammatory potential. The aim of present investigation was to evaluate the potential and possible mechanism of action of pinitol against lipopolysaccharide (LPS)‐induced pneumonia in the experimental animal model. Pneumonia was induced in Sprague‐Dawley rats by intratracheal administration of LPS (2 mg/kg). Animals were treated with either vehicle or dexamethasone or pinitol (5 or 10 or 20 mg/kg). Potential of pinitol against LPS‐induced pulmonary insult was assessed based on behavioral, biochemical, molecular, and ultrastructural studies. Intratracheal instillation of LPS induced significant (P < .05) inflammatory infiltration in bronchoalveolar lavage fluid (BALF) and lung tissue reflected by elevated pleural effusion volume, lung edema, BALF polymorphonuclear leukocytes count and lung myeloperoxidase levels, which was attenuated by pinitol (10 and 20 mg/kg) administration. Pinitol also markedly (P < .05) inhibited LPS‐induced alterations in electrocardiographic, hemodynamic changes, right ventricular, and lung function tests. The LPS‐induced downregulated nuclear factor erythroid 2–related factor 2 (Nrf‐2) and heme oxygenase‐1 (HO‐1), whereas upregulated transforming growth factor‐β (TGF‐β), tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3), and inducible nitric oxide synthase (iNOs) lung messenger RNA expressions were significantly (P < .05) inhibited by pinitol. Western blot analysis suggested pinitol markedly (P < .05) decreased nuclear factor‐κB (NF‐κB), inhibitor of nuclear factor κB (IkBα), toll‐like receptor 4 (TLR‐4), and cyclooxygenase‐II (COX‐II) protein expressions in the lung. These findings were further supported by histological and ultrastructural analyses of lung tissue that show pinitol significantly (P < .05) ameliorates LPS‐induced aberrations in lung tissue. In conclusion, pinitol attenuated LPS‐induced pneumonia via inhibition of TLR‐4 to downregulate the NF‐κB/IκBα signaling cascade and thus ameliorated the production of proinflammatory cytokines (TNF‐α, ILs, NLRP3, and TGF‐β), inflammatory mediators (COX‐II and iNOs) and elevated oxidative stress (Nrf‐2 and HO‐1).     

Trans‐repression of NFκB pathway mediated by PPARγ improves vascular endothelium insulin resistance

Previous study has shown that thiazolidinediones (TZDs) improved endothelium insulin resistance (IR) induced by high glucose concentration (HG)/hyperglycaemia through a PPARγ‐dependent‐NFκB trans‐repression mechanism. However, it is unclear, whether changes in PPARγ expression affect the endothelium IR and what the underlying mechanism is. In the present study, we aimed to address this issue. HG‐treated human umbilical vascular endothelial cells (HUVEC) were transfected by either PPARγ‐overexpressing (Ad‐PPARγ) or PPARγ‐shRNA‐containing (Ad‐PPARγ‐shRNA) adenoviral vectors. Likewise, the rats fed by high‐fat diet (HFD) were infected by intravenous administration of Ad‐PPARγ or Ad‐PPARγ‐shRNA. The levels of nitric oxide (NO), endothelin‐1 (ET‐1) and cytokines (TNFα, IL‐6, sICAM‐1 and sVCAM‐1) and the expression levels of PPARγ, eNOS, AKT, p‐AKT, IKKα/β and p‐IKKα/β and IκBα were examined; and the interaction between PPARγ and NFκB‐P65 as well as vascular function were evaluated. Our present results showed that overexpression of PPARγ notably increased the levels of NO, eNOS, p‐AKT and IκBα as well as the interaction of PPARγ and NFκB‐P65, and decreased the levels of ET‐1, p‐IKKα/β, TNFα, IL‐6, sICAM‐1 and sVCAM‐1. In contrast, down‐expression of PPARγ displayed the opposite effects. The results demonstrate that the overexpression of PPARγ improves while the down‐expression worsens the endothelium IR via a PPARγ‐mediated NFκB trans‐repression dependent manner. The findings suggest PPARγ is a potential therapeutic target for diabetic vascular complications.     

The deubiquitinase activity of A20 is dispensable for NF‐κB signaling

A20 has been suggested to limit NF‐κB activation by removing regulatory ubiquitin chains from ubiquitinated substrates. A20 is a ubiquitin‐editing enzyme that removes K63‐linked ubiquitin chains from adaptor proteins, such as RIP1, and then conjugates them to K48‐linked polyubiquitin chains to trigger proteasomal degradation. To determine the role of the deubiquitinase function of A20 in downregulating NF‐κB signaling, we have generated a knock‐in mouse that lacks the deubiquitinase function of A20 (A20‐OTU mice). These mice are normal and have no signs of inflammation, have normal proportions of B, T, dendritic, and myeloid cells, respond normally to LPS and TNF, and undergo normal NF‐κB activation. Our results thus indicate that the deubiquitinase activity of A20 is dispensable for normal NF‐κB signaling.     

Cerebral nitric oxide represses choroid plexus NFκB‐dependent gateway activity for leukocyte trafficking

Chronic neuroinflammation is evident in brain aging and neurodegenerative disorders and is often associated with excessive nitric oxide (NO) production within the central nervous system (CNS). Under such conditions, increased NO levels are observed at the choroid plexus (CP), an epithelial layer that forms the blood–cerebrospinal fluid barrier (BCSFB) and serves as a selective gateway for leukocyte entry to the CNS in homeostasis and following injury. Here, we hypothesized that elevated cerebral NO levels interfere with CP gateway activity. We found that induction of leukocyte trafficking determinants by the CP and sequential leukocyte entry to the CSF are dependent on the CP epithelial NFκB/p65 signaling pathway, which was inhibited upon exposure to NO. Examining the CP in 5XFAD transgenic mouse model of Alzheimer''s disease (AD-Tg) revealed impaired ability to mount an NFκB/p65-dependent response. Systemic administration of an NO scavenger in AD-Tg mice alleviated NFκB/p65 suppression at the CP and augmented its gateway activity. Together, our findings identify cerebral NO as a negative regulator of CP gateway activity for immune cell trafficking to the CNS.     

Central dopamine action modulates neuropeptide‐controlled appetite via the hypothalamic PI3K/NF‐κB‐dependent mechanism

Hypothalamic neuropeptides, including neuropeptide Y (NPY) and proopiomelanocortin (POMC), have been found to control the appetite‐suppressing effect of amphetamine (AMPH). In this study, we have examined whether dopamine receptor (DAR), phosphatidylinositol 3‐kinase (PI3K) and nuclear factor‐kappaB (NF‐κB) are involved in AMPH's action. We administered AMPH to rats once a day for 4 days and assessed and compared changes in hypothalamic NPY, melanocortin receptor 4 (MC4R), PI3K, pAkt and NF‐κB expression. We found that the inhibition of DAR increased NPY, but decreased MC4R, PI3K and NF‐κB expression, compared with AMPH‐treated rats. Moreover, MC4R, PI3K, pAkt and NF‐κB increased with the maximum response on Day 2, which was consistent with the response of feeding behavior, but was opposite to the expression of NPY. Furthermore, we found that the intracerebroventricular infusion of the PI3K inhibitor or NF‐κB antisense could attenuate AMPH‐induced anorexia, and partially reverse the expression of NPY, MC4R, PI3K, Akt and NF‐κB back toward a normal level. We, therefore, suggest that DAR–PI3K–NF‐κB signaling in the hypothalamus plays functional roles in the modulation of NPY and POMC neurotransmissions and in the control of AMPH‐evoked appetite suppression.     

Pannexin3 inhibits TNF‐α‐induced inflammatory response by suppressing NF‐κB signalling pathway in human dental pulp cells

Human dental pulp cells (HDPCs) play a crucial role in dental pulp inflammation. Pannexin 3 (Panx3), a member of Panxs (Pannexins), has been recently found to be involved in inflammation. However, the mechanism of Panx3 in human dental pulp inflammation remains unclear. In this study, the role of Panx3 in inflammatory response was firstly explored, and its potential mechanism was proposed. Immunohistochemical staining showed that Panx3 levels were diminished in inflamed human and rat dental pulp tissues. In vitro, Panx3 expression was significantly down‐regulated in HDPCs following a TNF‐α challenge in a concentration‐dependent way, which reached the lowest level at 10 ng/ml of TNF‐α. Such decrease could be reversed by MG132, a proteasome inhibitor. Unlike MG132, BAY 11‐7082, a NF‐κB inhibitor, even reinforced the inhibitory effect of TNF‐α. Quantitative real‐time PCR (qRT‐PCR) and enzyme‐linked immunosorbent assay (ELISA) were used to investigate the role of Panx3 in inflammatory response of HDPCs. TNF‐α‐induced pro‐inflammatory cytokines, interleukin (IL)‐1β and IL‐6, were significantly lessened when Panx3 was overexpressed in HDPCs. Conversely, Panx3 knockdown exacerbated the expression of pro‐inflammatory cytokines. Moreover, Western blot, dual‐luciferase reporter assay, immunofluorescence staining, qRT‐PCR and ELISA results showed that Panx3 participated in dental pulp inflammation in a NF‐κB‐dependent manner. These findings suggested that Panx3 has a defensive role in dental pulp inflammation, serving as a potential target to be exploited for the intervention of human dental pulp inflammation.     

Nitric oxide‐mediated inhibition of NFκB regulates hyperthermia‐induced apoptosis

Ascertaining the upstream regulatory mechanisms of hyperthermia‐induced apoptosis is important to understand the role of hyperthermia in combined modality cancer therapy. Accordingly, we investigated whether (i) hyperthermia‐induced apoptosis is mediated through the nitric oxide (NO) signaling pathway and (ii) inhibition of post‐translational modification of IκBα and down regulation of NFκB‐DNA binding activity is an intermediate step in NO‐dependent apoptosis in MCF‐7 breast cancer cells. For hyperthermia treatment, the cells were exposed to 43°C. Intracellular NO levels measured by the fluorescent intensity of DAF‐2A and iNOS expression by immunobloting revealed an increased level of iNOS dependent NO production after 43°C. Apoptosis measured by Annexin V expression and cell survival by clonogenic assay showed a 20% increase in apoptosis after 43°C treatments. EMSA analysis showed a dose‐dependent inhibition of NFκB‐DNA binding activity. The hyperthermia‐mediated inhibition of NFκB was persistent even after 48 h. Inhibition of NO by L ‐NAME rescued the NFκB‐DNA binding activity and inhibits heat‐induced apoptosis. Similarly, over‐expression of NFκB by transient transfection inhibits heat‐induced apoptosis. These results demonstrate that apoptosis upon hyperthermia exposure of MCF‐7 cells is regulated by NO‐mediated suppression of NFκB. J. Cell. Biochem. 106: 999–1009, 2009. © 2009 Wiley‐Liss, Inc.     

Propofol inhibits proliferation and induces neuroapoptosis of hippocampal neurons in vitro via downregulation of NF‐κB p65 and Bcl‐2 and upregulation of caspase‐3

Propofol is widely used in paediatric anaesthesia and intensive care unit because of its essentially short‐acting anaesthetic effect. Recent data have shown that propofol induced neurotoxicity in developing brain. However, the mechanisms are not extremely clear. To gain a better insight into the toxic effects of propofol on hippocampal neurons, we treated cells at the days in vitro 7 (DIV 7), which were prepared from Sprague–Dawley embryos at the 18th day of gestation, with propofol (0.1–1000 μM) for 3 h. A significant decrease in neuronal proliferation and a remarkable increase in neuroapoptosis were observed in DIV 7 hippocampal neurons as measured by 3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and apoptosis assay respectively. Moreover, propofol treatment decreased the nuclear factor kappaB (NF‐κB) p65 expression, which was accompanied by a reduction in B‐cell lymphoma 2 (Bcl‐2) mRNA and protein levels, increased caspase‐3 mRNA and activation of caspase‐3 protein. These results indicated that downregulation of NF‐κB p65 and Bcl‐2 were involved in the potential mechanisms of propofol‐induced neurotoxicity. This likely led to the caspase‐3 activation, triggered apoptosis and inhibited the neuronal growth and proliferation that we have observed in our in vitro systems. Copyright © 2014 John Wiley & Sons, Ltd.     

Petchiether A attenuates obstructive nephropathy by suppressing TGF‐β/Smad3 and NF‐κB signalling

Obstructive nephropathy is the end result of a variety of diseases that block drainage from the kidney(s). Transforming growth factor‐β1 (TGF‐β1)/Smad3‐driven renal fibrosis is the common pathogenesis of obstructive nephropathy. In this study, we identified petchiether A (petA), a novel small‐molecule meroterpenoid from Ganoderma, as a potential inhibitor of TGF‐β1‐induced Smad3 phosphorylation. The obstructive nephropathy was induced by unilateral ureteral obstruction (UUO) in mice. Mice received an intraperitoneal injection of petA/vehicle before and after UUO or sham operation. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin‐1β and tumour necrosis factor‐α) and reducing extracellular matrix deposition (α‐smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF‐κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down‐regulated the expression of the fibrotic marker collagen I in TGF‐β1‐treated renal epithelial cells. Further, we found that petA dose‐dependently suppressed Smad3‐responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF‐β/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF‐β/Smad3 signalling.