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An imbalance between oxidative stress and antioxidant activity plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke, a major risk factor of COPD, induces cellular oxidative stress, but levels of antioxidants such as heme oxygenase-1 (HO-1) are reduced in individuals with severe COPD. In this study, we evaluated the molecular mechanism of reduced HO-1 expression in human bronchial epithelial cells. We found that cigarette smoke extract (CSE) increases HO-1 levels via activation of NFE2-related factor 2 (Nrf2). However, pretreating cells with the protease neutrophil elastase (NE) suppressed the CSE-induced expression of HO-1 mRNA and protein. NE also decreased the sirtuin 1 (SIRT1) level, but did not inhibit CSE-induced nuclear translocation and DNA-binding activity of Nrf2. Transfection of cells with a Myc/His-tagged SIRT1 expression vector completely blocked the NE-mediated suppression of HO-1 expression. We further noted that the NE-induced down-regulation of SIRT1 was not due to decreased transcription or proteasomal/ lysosomal degradation or loss of solubility. Immunofluorescence staining revealed that NE enters the cell cytoplasm, and we observed that NE directly cleaved SIRT1 in vitro, indicating that SIRT1 levels are decreased via direct degradation by internalized NE. Of note, we observed decreased SIRT1 levels in NE-treated primary human bronchial epithelial cells and in lung homogenates from both smokers and patients with COPD. In conclusion, NE suppresses CSE-induced HO-1 expression by cleaving SIRT1. This finding indicates the importance of crosstalk between oxidative stress and protease responses in the pathogenesis of COPD. [ABSTRACT FROM AUTHOR]

Inflammation by IL-8-induced neutrophil recruitment and apoptosis of epithelial cells by decreased expression of VEGF have been suggested as one of the complicated pathogenic mechanisms of chronic obstructive pulmonary disease (COPD). The role of neutrophil elastase (NE) in the development of COPD is also well known. However, little is known about how they interact. The objective of this study was to elucidate the effect of NE on cigarette smoke extract (CSE)-induced IL-8 and VEGF production and its molecular mechanism in bronchial epithelial cells. CSE increased both IL-8 and VEGF production in human bronchial epithelial cells (BEAS-2B). Although NE significantly enhanced CSE-induced IL-8 production, it suppressed VEGF production. This differential regulation was not CSE-specific. The effect of NE on IL-8 production, but not VEGF, was ERK-dependent. Interestingly, in contrast to decreased VEGF protein expression, NE accelerated VEGF transcription by CSE, suggesting post-translational modification. When cells were incubated with purified NE, it was detected in the cytoplasm, suggesting the intracellular translocation of NE. Furthermore, NE fragmented recombinant human VEGF in vitro but not recombinant human IL-8. These results indicate that VEGF down-regulation is due to direct degradation by NE, which is translocated into cells. Similar to in vitro cell experiments, elastase treatment increased CSE-induced IL-8; however, it suppressed VEGF production in bronchoalveolar lavage fluid of CSE-treated mice. Moreover, elastase treatment enhanced CSE-induced emphysema in mice. Considering the actions of IL-8 and VEGF, our results suggest that NE contributes to the pathogenesis of COPD by enhancing inflammation and apoptosis. [ABSTRACT FROM AUTHOR]

Proteasome inhibitors (PIs) have been reported to induce apoptosis in many types of tumor. Their apoptotic activities have been suggested to be associated with the up-regulation of molecules implicated in pro-apoptotic cascades such as p53, p21Waf1, and p27Kip1. Moreover, the blocking of NF-κB nuclear translocation via the stabilization of IκB is an important mechanism of PI-induced apoptosis. However, we found that longterm incubation with PIs (PS-341 or MG132) increased NF-κB-regulated gene expression such as COX-2, cIAP2, XIAP, and IL-8 in a dose- and time-dependent manner, which was mediated by phosphorylation of IκBα and its subsequent degradation via the alternative route, lysosome. Overexpression of the IκBα superrepressor (IκBα-SR) blocked PI-induced NF-κB activation. Treatment with lysosomal inhibitors (ammonium chloride or chloroquine) or inhibitors of cathepsins (Z-FF-FMK or Z-FAFMK) or knock-down of LC3B expression by siRNAs suppressed PI-induced IκBα degradation. Furthermore, we found that both IKK-dependent and IKK-independent pathways were required for PI-induced IκBα degradation. Pretreatment with IKKβ specific inhibitor, SC-514, partially suppressed IκBα degradation and IL-8 production by PIs. Blockade of IKK activity using insolubilization by heat shock (HS) and knock-down by siRNAs for IKKβ only delayed IκBα degradation up to 8 h after treatment with PIs. In addition, PIs induced Akt-dependent inactivation of GSK-3β. Inactive GSK-3β accelerated PI-induced IκBα degradation. Overexpression of active GSK-3β (S9A) or knock-down of GSK-3βdelayed PI-induced IκBα degradation. Collectively, our data demonstrate that long-term incubation with PIs activates NF-κB, which is mediated by IκBα degradation via the lysosome in an IKK-dependent and IKK-independent manner. [ABSTRACT FROM AUTHOR]

The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during Gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. UponMDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) and nucleotide-binding domain, leucine-rich repeat (NLR) proteins are two major forms of innate immune receptors that trigger inflammatory responses by various biological mechanisms such as cytokine production, recruitment of inflammatory cells, or activation of adaptive immunity. Although the innate immune system is designed to fight against infectious pathogens, excessive activation of TLR or NLR signaling pathways may lead to unwarranted inflammation with hazardous outcomes, including septic shock or inflammatory diseases. As part of the search for effective therapeutics to regulate these responses, here we show that a novel aminosaccharide compound, named DFK1012, inhibits immune responses caused by TLR and NLR activation. Treatment with DFK1012, but not its derivatives DFK845 or DFK846, strongly inhibited pro-inflammatory cytokine production upon stimulation via either TLR or NLR proteins in macrophages. Importantly, we have not observed cytotoxicity in any range of its working concentration. Treatment with DFK1012 did not interfere with TLR- or NLR-induced activation of p38 and JNK, phosphorylation/degradation of I?B, and subsequent nuclear translocation of NF-?B subunit p65, suggesting that the inhibitory activity of DFK1012 is not due to the suppression of downstream signaling. Indeed, DFK1012 did not impair transcription of pro-inflammatory cytokine genes but rather promoted post-translational degradation of pro-inflammatory cytokines. Therefore, DFK1012 is a novel anti-inflammatory compound that drives proteolysis of proinflammatory cytokines induced by TLR and NLR stimulation. DFK1012 may represent a novel class of potential therapeutic agents aimed at the treatment of inflammatory disorders. [ABSTRACT FROM AUTHOR]

Objectives: To estimate the cost-effectiveness of switching patients with poorly controlled type 2 diabetes mellitus from human insulin (HI) to biphasic insulin aspart 30 (BIAsp 30) in South Korea. Methods: A published and validated diabetes computer simulation model (the IMS CORE Diabetes Model) was used to evaluate the long-term clinical and economic outcomes associated with switching to BIAsp 30, using treatment effects from the South Korean subgroup of the Physician's Routine Evaluation of Safety and Efficacy of NovoMix® 30 Therapy study and cost data collected through primary research. Outcomes included life expectancy, quality-adjusted life expectancy, incidence of complications, direct medical costs, and cost-effectiveness. Analyses were performed from a third-party payer perspective over a 30-year time horizon. Future costs and clinical benefits were discounted at 5% per annum. Extensive sensitivity analyses were performed. Results: Switching patients uncontrolled on HI to BIAsp 30 was projected to increase discounted mean life expectancy by 0.15 ± 0.18 years per patient (8.62 ± 0.13 years vs. 8.47 ± 0.13 years) and improve discounted mean quality-adjusted life expectancy by 0.30 ± 0.12 quality-adjusted life-years (QALYs) per patient (5.68 ± 0.09 QALYs vs. 5.38 ± 0.09 QALYs). Conversion to BIAsp 30 was associated with a mean increase in direct costs of South Korean Won (KRW) 1,777,323 ± 359,209 over patient lifetimes. BIAsp 30 was associated with an incremental cost-effectiveness ratio of KRW5,916,758 per QALY gained versus HI. Conclusion: Switching patients uncontrolled on HI to BIAsp 30 was projected to improve life expectancy and quality-adjusted life expectancy. This analysis suggests that BIAsp 30 could be a cost-effective treatment option in type 2 diabetes patients poorly controlled on HI in South Korea. [ABSTRACT FROM AUTHOR]

Heat shock (HS) activates mitogen-activated protein (MAP) kinases. Although prior exposure to nonlethal HS makes cells refractory to the lethal effect of a subsequent HS, it is unclear whether this also occurs in MAP kinase activation. This study was undertaken to evaluate the effect of a heat pretreatment on MAP kinase activation by a subsequent HS and to elucidate its possible mechanism. Preheating did not make BEAS-2B cells refractory to extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) activation by a second HS but accelerated their inactivation after HS. The rapid inactivation of ERK and JNK was dependent on de novo protein synthesis and associated with the up-regulation of heat shock protein 70 (HSP70). Moreover, the inhibition of phosphatase activity reversed this rapid inactivation. MAP kinase phosphatase-1 (MKP-1) expression was increased by HS, and the presence of its phosphorylated form (p-MKP-1) correlated with the observed rapid ERK and JNK inactivation. Blocking induction of p-MKP-1 with antisense MKP-1 oligonucleotides suppressed the rapid inactivation of ERK and JNK in preheated cells. HSP70 overexpression caused the early phosphorylation of MKP-1. Moreover, MPK-1 phosphorylation and the rapid inactivation of ERK were inhibited by blocking HSP70 induction in preheated cells. In addition, MKP-1 was insolubilized by HS, and HSP70 associated physically with MKP-1, suggesting that a chaperone effect of HSP70 might have caused the early phosphorylation of MKP-1. These results indicate that preheating accelerated MAP kinase inactivation after a second HS and that this is related to a HSP70-mediated increase in p-MKP-1. [ABSTRACT FROM AUTHOR]