Bestrophin 3 Ameliorates TNFα-Induced Inflammation by Inhibiting NF-κB Activation in Endothelial Cells

Increasing evidences have suggested vascular endothelial inflammatory processes are the initiator of atherosclerosis. Bestrophin 3 (Best-3) is involved in the regulation of cell proliferation, apoptosis and differentiation of a variety of physiological functions, but its function in cardiovascular system remains unclear. In this study, we investigated the effect of Best-3 on endothelial inflammation. We first demonstrated that Best-3 is expressed in endothelial cells and decreased after tumor necrosis factor-α (TNFα) challenge. Overexpression of Best-3 significantly attenuated TNFα-induced expression of adhesion molecules and chemokines, and subsequently inhibited the adhesion of monocytes to human umbilical vein endothelial cells (HUVECs). Conversely, knockdown of Best-3 with siRNA resulted in an enhancement on TNFα-induced expression of adhesion molecules and chemokines and adhesion of monocytes to HUVECs. Furthermore, overexpression of Best-3 with adenovirus dramatically ameliorated inflammatory response in TNFα-injected mice. Mechanistically, we found up-regulation of Best-3 inhibited TNFα-induced IKKβ and IκBα phosphorylation, IκBα degradation and NF-κB translocation. Our results demonstrated that Best-3 is an endogenous inhibitor of NF-κB signaling pathway in endothelial cells, suggesting that forced Best-3 expression may be a novel approach for the treatment of vascular inflammatory diseases.     

Effusanin E Suppresses Nasopharyngeal Carcinoma Cell Growth by Inhibiting NF-κB and COX-2 Signaling

Rabdosia serra is well known for its antibacterial, anti-inflammatory and antitumor activities, but no information has been available for the active compounds derived from this plant in inhibiting human nasopharyngeal carcinoma (NPC) cell growth. In this study, we isolated and purified a natural diterpenoid from Rabdosia serra and identified its chemical structure as effusanin E and elucidated its underlying mechanism of action in inhibiting NPC cell growth. Effusanin E significantly inhibited cell proliferation and induced apoptosis in NPC cells. Effusanin E also induced the cleavage of PARP, caspase-3 and -9 proteins and inhibited the nuclear translocation of p65 NF-κB proteins. Moreover, effusanin E abrogated the binding of NF-κB to the COX-2 promoter, thereby inhibiting the expression and promoter activity of COX-2. Pretreatment with a COX-2 or NF-κB-selective inhibitor (celecoxib or ammonium pyrrolidinedithiocarbamate) had an additive effect on the effusanin E-mediated inhibition of proliferation, while pretreatment with an activator of NF-κB/COX-2 (lipopolysaccharides) abrogated the effusanin E-mediated inhibition of proliferation. Effusanin E also significantly suppressed tumor growth in a xenograft mouse model without obvious toxicity, furthermore, the expression of p50 NF-κB and COX-2 were down-regulated in the tumors of nude mice. These data suggest that effusanin E suppresses p50/p65 proteins to down-regulate COX-2 expression, thereby inhibiting NPC cell growth. Our findings provide new insights into exploring effusanin E as a potential therapeutic compound for the treatment of human nasopharyngeal carcinoma.     

FK506 Attenuates the Inflammation in Rat Spinal Cord Injury by Inhibiting the Activation of NF-κB in Microglia Cells

FK-506 (Tacrolimus) is a very commonly used immunomodulatory agent that plays important roles in modulating the calcium-dependent phosphoserine–phosphothreonine protein phosphatase calcineurin and thus inhibits calcineurin-mediated secondary neuronal damage. The biological function of FK-506 in the spinal cord has not been fully elucidated. To clarify the anti-inflammatory action of FK-506 in spinal cord injury (SCI), we performed an acute spinal cord contusion injury model in adult rats and hypoxia-treated primary spinal cord microglia cultures. This work studied the activation of NF-κB and proinflammatory cytokine (TNF-a, IL-1b, and IL-6) expression. ELISA and q-PCR analysis revealed that TNF-a, IL-1b, and IL-6 levels significantly increased 3 days after spinal cord contusion and decreased after 14 days, accompanied by the increased activation of NF-κB. This increase was reversed by an FK-506 treatment. Double immunofluorescence labeling suggested that NF-κB activation was especially prominent in microglia. Immunohistochemistry confirmed no alteration in the number of microglia. Moreover, the results in hypoxia-treated primary spinal cord microglia confirmed the effect of FK-506 on TNF-a, IL-1b, and IL-6 expression and NF-κB activation. These findings suggest that FK-506 may be involved in microglial activation after SCI.     

Activation of Mesenchymal Stem Cells by Macrophages Prompts Human Gastric Cancer Growth through NF-κB Pathway

Accumulating evidence indicate that macrophages activate mesenchymal stem cells (MSCs) to acquire pro-inflammatory phenotype. However, the role of MSCs activated by macrophages in gastric cancer remains largely unknown. In this study, we found that MSCs were activated by macrophages to produce increased levels of inflammatory cytokines. Cell colony formation and transwell migration assays revealed that supernatants from the activated MSCs could promote both gastric epithelial cell and gastric cancer cell proliferation and migration. In addition, the expression of epithelial-mesenchymal transition (EMT), angiogenesis, and stemness-related genes was increased in activated MSCs. The phosphorylated forms of NF-κB, ERK and STAT3 in gastric cells were increased by active MSCs. Inhibition of NF-κB activation by PDTC blocked the effect of activated MSCs on gastric cancer cells. Co-injection of activated MSCs with gastric cancer cells could accelerate gastric cancer growth. Moreover, human peripheral blood monocytes derived macrophages also activated MSCs to prompt gastric cancer cell proliferation and migration. Taken together, our findings suggest that MSCs activated by macrophage acquire pro-inflammatory phenotype and prompt gastric cancer growth in an NF-κB-dependent manner, which provides new evidence for the modulation of MSCs by tumor microenvironment and further insight to the role of stromal cells in gastric carcinogenesis and cancer progression.     

The Nuclear Factor NF-��B Pathway in Inflammation

The nuclear factor NF-κB pathway has long been considered a prototypical proinflammatory signaling pathway, largely based on the role of NF-κB in the expression of proinflammatory genes including cytokines, chemokines, and adhesion molecules. In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-κB in inflammation that suggest both pro- and anti-inflammatory roles for this pathway. NF-κB has long been considered the “holy grail” as a target for new anti-inflammatory drugs; however, these recent studies suggest this pathway may prove a difficult target in the treatment of chronic disease. In this article, we discuss the role of NF-κB in inflammation in light of these recent studies.NF-κB has long been considered a prototypical proinflammatory signaling pathway, largely based on the activation of NF-κB by proinflammatory cytokines such as interleukin 1 (IL-1) and tumor necrosis factor α (TNFα), and the role of NF-κB in the expression of other proinflammatory genes including cytokines, chemokines, and adhesion molecules, which has been extensively reviewed elsewhere. But inflammation is a complex physiological process and the role of NF-κB in the inflammatory response cannot be extrapolated from in vitro studies. In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-κB pathway in inflammation.     

NF-��B in the Immune Response of Drosophila

The nuclear factor κB (NF-κB) pathways play a major role in Drosophila host defense. Two recognition and signaling cascades control this immune response. The Toll pathway is activated by Gram-positive bacteria and by fungi, whereas the immune deficiency (Imd) pathway responds to Gram-negative bacterial infection. The basic mechanisms of recognition of these various types of microbial infections by the adult fly are now globally understood. Even though some elements are missing in the intracellular pathways, numerous proteins and interactions have been identified. In this article, we present a general picture of the immune functions of NF-κB in Drosophila with all the partners involved in recognition and in the signaling cascades.The paramount roles of NF-κB family members in Drosophila development and host defense are now relatively well established and have been the subject of several in-depth reviews in recent years, including some from this laboratory (e.g., Hoffmann 2003; Minakhina and Steward 2006; Ferrandon et al. 2007; Lemaitre and Hoffmann 2007; Aggarwal and Silverman 2008). To avoid excessive duplication, we limit this text to the general picture that has evolved over nearly two decades—since the initial demonstration that the dorsal gene plays a role in dorsoventral patterning in embryogenesis of Drosophila and that it encodes a member of the NF-κB family of inducible transactivators (Nüsslein-Volhard et al. 1980; Steward 1987; Roth et al. 1989). In the early nineties, it became apparent that NF-κB also plays a role in the antimicrobial host defense of Drosophila (Engström et al. 1993; Ip et al. 1993; Kappler et al. 1993; Reichhart et al. 1993). We focus in this article on the immune functions of NF-κB and refer the reader to recent reviews for the roles of NF-κB in development (Roth 2003; Brennan and Anderson 2004; Moussian and Roth 2005; Minakhina and Steward 2006).The Drosophila genome codes for three NF-κB family members (Fig. 1). Dorsal and DIF (for dorsal-related immunity factor) are 70 kDa proteins, with a typical Rel homology domain, which is 45% identical to that of the mammalian counterparts c-Rel, Rel A, and Rel B. Dorsal and DIF lie some 10 kbp apart on the second chromosome and probably arose from a recent duplication (Meng et al. 1999). Both proteins are retained in the cytoplasm by binding to the same 54-kDa inhibitor protein Cactus, which is homologous to mammalian IκBs (Schüpbach and Wieshaus 1989; Geisler et al. 1992). The single Drosophila Cactus gene is closest to mammalian IκBα (Huguet et al. 1997). The third member of the family in Drosophila, Relish, is a 100-kDa protein with an amino-terminal Rel domain and a carboxy-terminal extension with typical ankyrin repeats, as found in Cactus and mammalian IκBs. Relish is similar to mammalian p100 and p105 and its activation requires proteolytic cleavage as in the case for these mammalian counterparts (reviewed in Hultmark 2003).Open in a separate windowFigure 1.The NF-κB and IκB proteins in Drosophila. The length in amino acids is indicated by numbers. REL, Rel-homology domain; NLS, nuclear localization sequence; PEST, proline, glutamic acid, serine, and threonine-rich segment; Ac, acidic domain.Put in simple terms, NF-κB family members function in the host defense of Drosophila to control the expression of genes encoding immune-responsive peptides and proteins. Prominent among the induced genes are those encoding peptides with direct antimicrobial activity. To exert this function, Dorsal and DIF are translocated to the nucleus following stimulus-induced degradation of the inhibitor Cactus, whereas Relish requires stimulus-induced proteolytic cleavage for nuclear translocation of its amino-terminal Rel domain. This paradigm is similar to that observed in mammalian immunity. Again, for the sake of simplicity, we may say that the stimulus-induced degradation of Cactus, and the concomitant release of Dorsal or DIF, is primarily observed during Gram-positive bacterial and fungal infections and mediated by the Toll signaling pathway. In contrast, stimulus-induced proteolytic cleavage of Relish, and concomitant nuclear translocation of its amino-terminal Rel domain, is the hallmark of the response to Gram-negative bacterial infection and mediated by the Imd signaling pathway. Whether these pathways are also involved in the multifaceted defense against viruses remains an open question (Zambon et al. 2005). The Toll pathway was further shown to be involved in hematopoiesis of flies (Qiu et al. 1998). Of note, the Cactus-NF-κB module also plays a central role in the elimination of Plasmodium parasites in infected mosquitoes (Frolet et al. 2006). In the following, we review our information of the two established signaling pathways, Toll and Imd, which lead to gene reprogramming through NF-κB in response to bacterial and fungal infections. We first consider the upstream mechanisms that mediate the recognition of infection and allow for a certain level of discrimination between invading microorganisms. Gene reprogramming in this context is best illustrated by the induction of the antimicrobial peptide genes, which serve as the most convenient readouts of the antimicrobial defense of Drosophila (see Samakovlis et al. 1990; Reichhart et al. 1992; Ferrandon et al. 1998). Flies produce at least seven families of mostly cationic, small-sized, membrane-active peptides, with spectra variously directed against Gram-positive (defensins) and Gram-negative (diptericins, attacins, and drosocin) bacteria, and against fungi (drosomycins and metchnikowins), or with overlapping spectra (cecropins) (reviewed in Bulet et al. 1999; Hetru et al. 2003). The primary site of biosynthesis of these peptides is the fat body, a functional equivalent of the mammalian liver. Blood cells also participate in the production of antimicrobial peptides. As a rule, these molecules are secreted into the hemolymph where they reach remarkably high concentrations to oppose invading microorganisms (Hetru et al. 2003). This facet of the antimicrobial host defense is generally referred to as systemic immune response. Of note, the gut and the tracheae also produce antimicrobial peptides in response to microbes (see Tzou et al. 2000; Onfelt Tingvall et al. 2001; Liehl et al. 2006; Nehme et al. 2007).During infection, the Toll and Imd pathways control the expression of hundreds of genes. In addition to the antimicrobial peptides, these genes encode proteases, putative cytokines, cytoskeletal proteins, and many peptides and proteins whose function in the host defense are still not understood (De Gregorio et al. 2001; Irving et al. 2001).