Insulin inhibits hepatocyte iNOS expression induced by cytokines by an Akt-dependent mechanism

Hepatocyte inducible nitric oxide synthese (iNOS) expression is a tightly controlled pathway that mediates hepatic inflammation and hepatocyte injury in a variety of disease states. We have shown that cyclic adenosine monophosphate (cAMP) regulates cytokine-induced hepatocyte iNOS expression through mechanisms that involve protein kinase B/Akt. We hypothesized that insulin, which activates Akt signaling in hepatocytes, as well as signaling through p38 and MAPK p42/p44, would regulate iNOS expression during inflammation. In primary rat hepatocytes, insulin inhibited cytokine-stimulated nitrite accumulation and iNOS expression in a dose-dependent manner. Inhibition of MAPK p42/p44 with PD98059 had no effect on iNOS activation, whereas SB203580 to block p38 reversed insulin's inhibitory effect. However, insulin did not increase p38 activation and inhibition of p38 signaling with a dominant negative p38 plasmid had no effect on cytokine- or insulin-mediated effects on iNOS. We found that SB203580 blocked insulin-induced Akt activation. Inhibition of Akt signaling with LY294002 or a dominant negative Akt plasmid increased cytokine-stimulated nitrite production and iNOS protein expression and blocked the inhibitory effects of insulin. NF-κB induces iNOS expression and can be regulated by Akt, but insulin had no effect on cytokine-mediated IκBα levels or NF-κB p65 translocation. Our data demonstrate that insulin inhibits cytokine-stimulated hepatocyte iNOS expression and does so through effects on Akt-mediated signaling.     

Resveratrol preconditioning modulates inflammatory response in the rat hippocampus following global cerebral ischemia

Considerable evidence has been accumulated to suggests that blocking the inflammatory reaction promotes neuroprotection and shows therapeutic potential for clinical treatment of ischemic brain injury. Consequently, anti-inflammatory therapies are being explored for prevention and treatment of these diseases. Induction of brain tolerance against ischemia by pretreatment with resveratrol has been found to influence expression of different molecules. It remains unclear, however, whether and how resveratrol preconditioning changes expression of inflammatory mediators after subsequent global cerebral ischemia/reperfusion (I/R). Therefore, we investigated the effect of resveratrol pretreatment on NF-κB inflammatory cascade, COX-2, iNOS and JNK levels in experimental I/R. Adult male rats were subjected to 10min of four-vessel occlusion and sacrificed at selected post-ischemic time points. Resveratrol (30mg/kg) pretreatment was injected intraperitoneally 7days prior to I/R induction. We found that resveratrol treatment before insult remarkably reduced astroglial and microglial activation at 7days after I/R. It greatly attenuated I/R-induced NF-κB and JNK activation with decreased COX-2 and iNOS production. In conclusion, the neuroprotection of resveratrol preconditioning may be due in part to the suppression of the inflammatory response via regulation of NF-κB, COX-2 and iNOS induced by I/R. JNK was also suggested to play a protective role through in neuroprotection of resveratrol, which may also be contributing to reduction in neuroinflammation. The study adds to a growing literature that resveratrol can have important anti-inflammatory actions in the brain.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

Constitutive NF-kappaB activation confers interleukin 6 (IL6) independence and resistance to dexamethasone and Janus kinase inhibitor INCB018424 in murine plasmacytoma cells

Myeloma cells are dependent on IL6 for their survival and proliferation during the early stages of disease, and independence from IL6 is associated with disease progression. The role of the NF-κB pathway in the IL6-independent growth of myeloma cells has not been studied. Because human herpesvirus 8-encoded K13 selectively activates the NF-κB pathway, we have used it as a molecular tool to examine the ability of the NF-κB pathway to confer IL6 independence on murine plasmacytomas. We demonstrated that ectopic expression of K13, but not its NF-κB-defective mutant or a structural homolog, protected plasmacytomas against IL6 withdrawal-induced apoptosis and resulted in emergence of IL6-independent clones that could proliferate long-term in vitro in the absence of IL6 and form abdominal plasmacytomas with visceral involvement when injected intraperitoneally into syngeneic mice. These IL6-independent clones were dependent on NF-κB activity for their survival and proliferation but were resistant to dexamethasone and INCB018424, a selective Janus kinase 1/2 inhibitor. Ectopic expression of human T cell leukemia virus 1-encoded Tax protein, which resembles K13 in inducing constitutive NF-κB activation, similarly protected plasmacytoma cells against IL6 withdrawal-induced apoptosis. Although K13 is known to up-regulate IL6 gene expression, its protective effect was not due to induction of endogenous IL6 production but instead was associated with sustained expression of several antiapoptotic members of the Bcl2 family upon IL6 withdrawal. Collectively, these results demonstrate that NF-κB activation cannot only promote the emergence of IL6 independence during myeloma progression but can also confer resistance to dexamethasone and INCB018424.     

Expression of TNFAIP3 in intestinal epithelial cells protects from DSS- but not TNBS-induced colitis

Intestinal epithelial cells (IEC) maintain gastrointestinal homeostasis by providing a physical and functional barrier between the intestinal lumen and underlying mucosal immune system. The activation of NF-κB and prevention of apoptosis in IEC are required to maintain the intestinal barrier and prevent colitis. How NF-κB activation in IEC prevents colitis is not fully understood. TNFα-induced protein 3 (TNFAIP3) is a NF-κB-induced gene that acts in a negative-feedback loop to inhibit NF-κB activation and also to inhibit apoptosis; therefore, we investigated whether TNFAIP3 expression in the intestinal epithelium impacts susceptibility of mice to colitis. Transgenic mice expressing TNFAIP3 in IEC (villin-TNFAIP3 Tg mice) were exposed to dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the severity and characteristics of mucosal inflammation and barrier function were compared with wild-type mice. Villin-TNFAIP3 Tg mice were protected from DSS-induced colitis and displayed reduced production of NF-κB-dependent inflammatory cytokines. Villin-TNFAIP3 Tg mice were also protected from DSS-induced increases in intestinal permeability and induction of IEC death. Villin-TNFAIP3 Tg mice were not protected from colitis induced by TNBS. These results indicate that TNFAIP3 expression in IEC prevents colitis involving DSS-induced IEC death, but not colitis driven by T cell-mediated inflammation. As TNFAIP3 inhibits NF-κB activation and IEC death, expression of TNFAIP3 in IEC may provide an avenue to inhibit IEC NF-κB activation without inducing IEC death and inflammation.     

Inhibition of NF-κB activation by diethylcarbamazine prevents alcohol-induced liver injury in C57BL/6 mice

Induction of NF-κB-mediated gene expression has been identified in the pathogenesis of alcoholic liver disease (ALD). Diethylcarbamazine (DEC) is a piperazine derivative drug with anti-inflammatory properties. The present study was designed to evaluate the effect of DEC on NF-κB pathways in mice undergoing alcoholism induced hepatic inflammation. Forty male C57BL/6 mice were divided equally into four groups: control group (C); DEC-treated group, which received 50 mg/kg (DEC50); alcoholic group (EtOH), submitted to chronic alcohol consumption and the alcohol-DEC treated group (EtOH50), submitted to chronic alcoholism consumption plus DEC treatment. Histological analysis of the alcoholic group showed evident hepatocellular damage which was reduced in EtOH50 group. Immunohistochemistry and western blot results showed elevated expression of inflammatory markers such as MDA, TNF-α, IL-1β, COX-2 and iNOS in hepatocytes of EtOH group. However, low immunopositivity for these markers was detected following DEC treatment. In the EtOH group the activation of NF-κB was observed by an increase in the expression of both NF-κB and pNF-κB in hepatocytes. This expression was significantly reduced in livers of EtOH50 group. Protein expression of Iκβα was measured to determine whether activation of NF-κB might be the result of Iκβα degradation. It was observed that expression of this protein was low in EtOH group, while animals treated with DEC had a high expression of Iκβα. The results of the present study indicate that DEC alleviates alcoholic liver injury, in part by the inhibiting activation of NF-κB and by suppressing the induction of NF-κB-dependent genes.     

NF-κB essential modulator (NEMO) interaction with linear and lys-63 ubiquitin chains contributes to NF-κB activation

The IκB kinase (IKK) complex acts as a gatekeeper of canonical NF-κB signaling in response to upstream stimulation. IKK activation requires sensing of ubiquitin chains by the essential IKK regulatory subunit IKKγ/NEMO. However, it has remained enigmatic whether NEMO binding to Lys-63-linked or linear ubiquitin chains is critical for triggering IKK activation. We show here that the NEMO C terminus, comprising the ubiquitin binding region and a zinc finger, has a high preference for binding to linear ubiquitin chains. However, immobilization of NEMO, which may be reminiscent of cellular oligomerization, facilitates the interaction with Lys-63 ubiquitin chains. Moreover, selective mutations in NEMO that abolish association with linear ubiquitin but do not affect binding to Lys-63 ubiquitin are only partially compromising NF-κB signaling in response to TNFα stimulation in fibroblasts and T cells. In line with this, TNFα-triggered expression of NF-κB target genes and induction of apoptosis was partially compromised by NEMO mutations that selectively impair the binding to linear ubiquitin chains. Thus, in vivo NEMO interaction with linear and Lys-63 ubiquitin chains is required for optimal IKK activation, suggesting that both type of chains are cooperating in triggering canonical NF-κB signaling.     

Selective abrogation of BiP/GRP78 blunts activation of NF-κB through the ATF6 branch of the UPR: involvement of C/EBPβ and mTOR-dependent dephosphorylation of Akt

Subtilase cytotoxin (SubAB) that selectively cleaves BiP/GRP78 triggers the unfolded protein response (UPR) and protects mice from endotoxic lethality and collagen arthritis. We found that pretreatment of cells with SubAB suppressed tumor necrosis alpha (TNF-α)-induced activation of NF-κB and NF-κB-dependent chemokine expression. To elucidate underlying mechanisms, the involvement of C/EBP and Akt, putative regulators of NF-κB, was investigated. Among members of the C/EBP family, SubAB preferentially induced C/EBPβ. Overexpression of C/EBPβ suppressed TNF-α-induced NF-κB activation, and knockdown of C/EBPβ attenuated the suppressive effect of SubAB on NF-κB. We identified that the ATF6 branch of the UPR plays a crucial role in the induction of C/EBPβ. In addition to this effect, SubAB depressed basal and TNF-α-induced phosphorylation of Akt via the UPR. It was mediated by the induction of ATF6 and consequent activation of mTOR that dephosphorylated Akt. Inhibition of Akt attenuated activation of NF-κB by TNF-α, suggesting that the mTOR-Akt pathway is another target for SubAB-initiated, UPR-mediated NF-κB suppression. These results elucidated that SubAB blunts activation of NF-κB through ATF6-dependent mechanisms, i.e., preferential induction of C/EBPβ and mTOR-dependent dephosphorylation of Akt.     

NF-kappaB-mediated modulation of inducible nitric oxide synthase activity controls induction of the Epstein-Barr virus productive cycle by transforming growth factor beta 1

Transforming growth factor beta 1 (TGF-β1) signal transduction has been implicated in many second-messenger pathways, including the NF-κB pathway. We provide evidence of a novel TGF-β1-mediated pathway that leads to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, which in turn induces expression of an Epstein-Barr virus (EBV) protein, ZEBRA, that is responsible for the induction of the viral lytic cycle. This pathway includes two unexpected steps, both of which are required to control ERK 1/2 phosphorylation: first, a quick and transient activation of NF-κB, and second, downregulation of inducible nitric oxide synthase (iNOS) activity that requires the participation of NF-κB activity. Although necessary, NF-κB alone is not sufficient to produce downregulation of iNOS, suggesting that another uncharacterized event(s) is involved in this pathway. Dissection of the steps involved in the switch from the EBV latent cycle to the lytic cycle will be important to understand how virus-host relationships modulate the innate immune system.