Del-1 Plays a Protective Role against COPD Development by Inhibiting Inflammation and Apoptosis.

Neutrophilic inflammation is a prominent feature of chronic obstructive pulmonary disease (COPD). Developmental endothelial locus-1 (Del-1) has been reported to limit excessive neutrophilic inflammation by inhibiting neutrophil adhesion to the vascular endothelial cells. However, the effects of Del-1 in COPD are not known. We investigated the role of Del-1 in the pathogenesis of COPD. Del-1 protein expression was decreased in the lungs of COPD patients, especially in epithelial cells and alveolar macrophages. In contrast to human lung tissue, Del-1 expression was upregulated in lung tissue from mice treated with cigarette smoke extracts (CSE). Overexpression of Del-1 significantly suppressed IL-8 release and apoptosis in CSE-treated epithelial cells. In contrast, knockdown of Del-1 enhanced IL-8 release and apoptosis. In macrophages, overexpression of Del-1 significantly suppressed inflammatory cytokine release, and knockdown of Del-1 enhanced it. This anti-inflammatory effect was mediated by inhibiting the phosphorylation and acetylation of NF-κB p65. Nuclear factor erythroid 2-related factor 2 (Nrf2) activators, such as quercetin, resveratrol, and sulforaphane, increased Del-1 in both cell types. These results suggest that Del-1, mediated by Nrf2, plays a protective role against the pathogenesis of COPD, at least in part through anti-inflammatory and anti-apoptotic effects.

Tetrahedral DNA nanostructures enhance transcription isothermal amplification for multiplex detection of non-coding RNAs.

This study introduces an innovative detection system for multiple cancer biomarkers, employing transcription isothermal amplification methods in conjunction with a tetrahedral DNA nanostructure (TDN). We demonstrate that TDN enhances various transcription isothermal amplification methods by placing DNA probes in proximity. Notably, the TDN-enhanced split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR) system stands out for its optimal performance and operational simplicity, especially in identifying non-coding RNAs such as microRNAs and long non-coding RNAs (lncRNAs). Multiplex detection of lncRNAs was also achieved by generating distinct light-up RNA aptamers, each emitting unique fluorescence signals. The system effectively identified the target lncRNAs, demonstrating high sensitivity and selectivity in both cell lines and clinical samples. The system, utilizing the single enzyme T7 RNA polymerase, can be easily tailored for alternative targets by substituting target-specific sequences in DNA probes and seamlessly integrated with other isothermal amplification methods for greater sensitivity and accuracy in the detection of multiple cancer biomarkers.

Analysis of PM2.5 inorganic and organic constituents to resolve contributing sources in Seoul, South Korea and Beijing, China and their possible associations with cytokine IL-8.

China and South Korea are the most polluted countries in East Asia due to significant urbanization and extensive industrial activities. As neighboring countries, collaborative management plans to maximize public health in both countries can be helpful in reducing transboundary air pollution. To support such planning, PM2.5 inorganic and organic species were determined in simultaneously collected PM2.5 integrated filters. The resulting data were used as inputs to positive matrix factorization, which identified nine sources at the ambient air monitoring sites in both sites. Secondary nitrate, secondary sulfate/oil combustion, soil, mobile, incinerator, biomass burning, and secondary organic carbon (SOC) were found to be sources at both sampling sites. Industry I and II were only identified in Seoul, whereas combustion and road dust sources were only identified in Beijing. A subset of samples was selected for exposure assessment. The expression levels of IL-8 were significantly higher in Beijing (167.7 pg/mL) than in Seoul (72.7 pg/mL). The associations between the PM2.5 chemical constituents and its contributing sources with PM2.5-induced inflammatory cytokine (interleukin-8, IL-8) levels in human bronchial epithelial cells were investigated. For Seoul, the soil followed by the secondary nitrate and the biomass burning showed increase with IL-8 production. However, for the Beijing, the secondary nitrate exhibited the highest association with IL-8 production and SOC and biomass burning showed modest increase with IL-8. As one of the highest contributing sources in both cities, secondary nitrate showed an association with IL-8 production. The soil source having the strongest association with IL-8 production was found only for Seoul, whereas SOC showed a modest association only for Beijing. This study can provide the scientific basis for identifying the sources to be prioritized for control to provide effective mitigation of particulate air pollution in each city and thereby improve public health.

Machine Learning-Based Proteomics Reveals Ferroptosis in COPD Patient-Derived Airway Epithelial Cells Upon Smoking Exposure.

BACKGROUND: Proteomics and genomics studies have contributed to understanding the pathogenesis of chronic obstructive pulmonary disease (COPD), but previous studies have limitations. Here, using a machine learning (ML) algorithm, we attempted to identify pathways in cultured bronchial epithelial cells of COPD patients that were significantly affected when the cells were exposed to a cigarette smoke extract (CSE). METHODS: Small airway epithelial cells were collected from patients with COPD and those without COPD who underwent bronchoscopy. After expansion through primary cell culture, the cells were treated with or without CSEs, and the proteomics of the cells were analyzed by mass spectrometry. ML-based feature selection was used to determine the most distinctive patterns in the proteomes of COPD and non-COPD cells after exposure to smoke extract. Publicly available single-cell RNA sequencing data from patients with COPD (GSE136831) were used to analyze and validate our findings. RESULTS: Five patients with COPD and five without COPD were enrolled, and 7,953 proteins were detected. Ferroptosis was enriched in both COPD and non-COPD epithelial cells after their exposure to smoke extract. However, the ML-based analysis identified ferroptosis as the most dramatically different response between COPD and non-COPD epithelial cells, adjusted P value = 4.172 × 10-6, showing that epithelial cells from COPD patients are particularly vulnerable to the effects of smoke. Single-cell RNA sequencing data showed that in cells from COPD patients, ferroptosis is enriched in basal, goblet, and club cells in COPD but not in other cell types. CONCLUSION: Our ML-based feature selection from proteomic data reveals ferroptosis to be the most distinctive feature of cultured COPD epithelial cells compared to non-COPD epithelial cells upon exposure to smoke extract.

Downregulation of Lysosome-Associated Membrane Protein-2A Contributes to the Pathogenesis of COPD.

Background: Macroautophagy plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD), but the role of chaperone-mediated autophagy (CMA) has not been investigated. We investigated if and how CMA is involved in the pathogenesis of COPD. Methods: We measured the level of lysosome-associated membrane protein-2A (LAMP-2A), which is a critical component of CMA that functions as a receptor for cytosolic substrate proteins, in total lung tissues and primary human bronchial epithelial cells (HBECs) from healthy never smokers, smokers, and COPD patients. We assessed the effects of LAMP-2A knock-down on cigarette smoke extract (CSE)-induced aging, cell cycle arrest, and apoptosis in BEAS-2B cells and the expression levels of apoptosis hallmarks in primary HBECs and lung tissue sections. Results: We found that the protein levels of LAMP-2A in lung homogenates and primary HBECs from smokers and COPD patients were lower than those from never smokers. In addition, its level in primary HBECs was negatively correlated with years of smoking. CSE caused degradation of LAMP-2A protein via the lysosomal pathway by activating macroautophagy. Knock-down of LAMP-2A markedly enhanced CSE-induced expression of senescence markers such as p16, p21, p27, and p53. G2/M cell cycle arrest, up-regulation of cyclin B1, and apoptosis in BEAS-2B cells. Apoptosis was increased in CSE-treated primary HBECs and in lung tissues from smokers and COPD patients. Conclusion: Cigarette smoke-induced down-regulation of LAMP-2A is involved in acceleration of aging and apoptosis of lung epithelial cells, which might at least partially contribute to COPD pathogenesis.

Cereblon Deficiency Contributes to the Development of Elastase-Induced Emphysema by Enhancing NF-κB Activation.

Cereblon (CRBN) has been shown to play an essential role in regulating inflammatory response and endoplasmic reticulum stress, thus mediating the development of various diseases. However, little is known about the roles of CRBN in chronic obstructive pulmonary disease (COPD) pathogenesis. We found that the protein levels of CRBN in lung homogenates from patients with COPD were lower than those from never smokers and smokers. The CRBN protein level was positively correlated with the forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC). To investigate the role of CRBN in modulating elastase-induced emphysema, we used Crbn knockout (KO) mice. Elastase-induced emphysematous changes were significantly aggravated in Crbn KO mice. Neutrophil infiltration, lung cell injury, and protein leakage into the bronchoalveolar space were more severe in Crbn KO mice than in wild-type (WT) mice. Furthermore, Crbn KO resulted in the elevated release of neutrophilic chemokines and inflammatory cytokines in lung epithelial cells and macrophages. The transcriptional activity of nuclear factor-κB (NF-κB) was significantly increased in Crbn knocked-down cells. In conclusion, Crbn deficiency might be involved in the development of emphysema by enhancing NF-κB activation, suggesting that targeting CRBN might be an effective therapeutic approach for the treatment of COPD.

T7 Endonuclease I-mediated voltammetric detection of KRAS mutation coupled with horseradish peroxidase for signal amplification.

Rapid and selective sensing of KRAS gene mutation which plays a crucial role in the development of colorectal, pancreatic, and lung cancers is of great significance in the early diagnosis of cancers. In the current study, we developed a simple electrochemical biosensor by differential pulse voltammetry technique for the specific detection of KRAS mutation that uses the mismatch-specific cleavage activity of T7-Endonuclease I (T7EI) coupled with horseradish peroxidase (HRP) to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) substrate in the presence of hydrogen peroxide (H2O2). In addition, we synthesized the nanocomposite composed of multi-walled carbon nanotube/chitosan-ionic liquid/gold nanoparticles (MWCNT/Chit-IL/AuNPs) on screen-printed carbon electrode surface to increase the electrode surface area and electrochemical signal. In principle, T7E1 enzyme recognized and cleaved the mismatched site formed by the presence of KRAS gene mutation, removing 5'-biotin of capture probes and subsequently reducing the differential pulse voltammetry signal compared to wild-type KRAS gene. With this proposed strategy, a limit of detection of 11.89 fM was achieved with a broad linear relationship from 100 fM to 1 µM and discriminated 0.1% of mutant genes from the wild-type target genes. This confirms that the developed biosensor is a potential platform for the detection of mutations in early disease diagnosis.

YPL-001 Shows Various Beneficial Effects against Cigarette Smoke Extract-Induced Emphysema Formation: Anti-Inflammatory, Anti-Oxidative, and Anti-Apoptotic Effects.

Inflammation, oxidative stress, and apoptosis are thought to be important causes of chronic obstructive pulmonary disease (COPD). We investigated the effect of YPL-001 (under phase 2a study, ClinicalTrials.gov identifier NCT02272634), a drug derived from Pseudolysimachion rotundum var. subintegrum, on cigarette smoke extract (CSE)-induced inflammation, the anti-oxidative pathway, and apoptosis in human lung epithelial cells and on CSE-induced emphysema in mice. YPL-001 suppressed CSE-induced expression of IL8 mRNA and protein. This was due to the reduction in NF-κB transcriptional activity by YPL-001, which resulted from the blockade of acetylation of the NF-κB subunit p65 (Lys310). Histone deacetylases (HDACs) prevent gene transcription by condensing the DNA structure and affecting NF-κB nuclear binding. YPL-001 alone increased HDAC2 activity and enhanced CSE-induced activation of HDAC2. YPL-001-induced suppression of NF-κB transcriptional activity might be caused by increased HDAC2 activity. YPL-001 increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression via both degradation of its inhibitory protein, Kelch-like ECH-associated protein 1, and an increase in de novo protein synthesis. YPL-001 increased the DNA binding activity of Nrf2. Consequently, YPL-001 upregulated the expression of Nrf2-targeted anti-oxidant genes such as NAD(P)H quinone dehydrogenase 1 and heme oxygenase 1. Moreover, YPL-001 significantly suppressed CSE-induced apoptotic cell death. In vivo study showed that CSE-induced emphysematous changes, neutrophilic inflammation, protein leakage into bronchoalveolar space, and lung cell apoptosis in mice were suppressed by YPL-001 treatment. Taken together, these results suggest that YPL-001 is a good therapeutic candidate for the treatment of COPD by blocking inflammation and apoptosis and activating the anti-oxidative pathway.

Developmental endothelial locus-1 as a potential biomarker for the incidence of acute exacerbation in patients with chronic obstructive pulmonary disease.

BACKGROUND: Despite the high disease burden of chronic obstructive pulmonary disease (COPD) and risk of acute COPD exacerbation, few COPD biomarkers are available. As developmental endothelial locus-1 (DEL-1) has been proposed to possess beneficial effects, including anti-inflammatory effects, we hypothesized that DEL-1 could be a blood biomarker for COPD. OBJECTIVE: To elucidate the role of plasma DEL-1 as a biomarker of COPD in terms of pathogenesis and for predicting acute exacerbation. METHODS: Cigarette smoke extract (CSE) or saline was intratracheally administered to wild-type (WT) and DEL-1 knockout (KO) C57BL/6 mice. Subsequently, lung sections were obtained to quantify the degree of emphysema using the mean linear intercept (MLI). Additionally, plasma DEL-1 levels were compared between COPD and non-COPD participants recruited in ongoing prospective cohorts. Using negative binomial regression analysis, the association between the plasma DEL-1 level and subsequent acute exacerbation risk was evaluated in patients with COPD. RESULTS: In the in vivo study, DEL-1 KO induced emphysema (KO saline vs. WT saline; P = 0.003) and augmented CSE-induced emphysema (KO CSE vs. WT CSE; P < 0.001) in 29 mice. Among 537 participants, patients with COPD presented plasma log (DEL-1) levels lower than non-COPD participants (P = 0.04), especially non-COPD never smokers (P = 0.019). During 1.2 ± 0.3 years, patients with COPD in the lowest quartile of Log(DEL-1) demonstrated an increased risk of subsequent acute exacerbation, compared with those in the highest quartile of Log(DEL-1) (adjusted incidence rate ratio, 3.64; 95% confidence interval, 1.03-12.9). CONCLUSION: Low DEL-1 levels are associated with COPD development and increased risk of subsequent COPD acute exacerbation. DEL-1 can be a useful biomarker in patients with COPD.

Three-Way Junction-Induced Isothermal Amplification with High Signal-to-Background Ratio for Detection of Pathogenic Bacteria.

The consumption of water and food contaminated by pathogens is a major cause of numerous diseases and deaths globally. To control pathogen contamination and reduce the risk of illness, a system is required that can quickly detect and monitor target pathogens. We developed a simple and reproducible strategy, termed three-way junction (3WJ)-induced transcription amplification, to detect target nucleic acids by rationally combining 3WJ-induced isothermal amplification with a light-up RNA aptamer. In principle, the presence of the target nucleic acid generates a large number of light-up RNA aptamers (Spinach aptamers) through strand displacement and transcription amplification for 2 h at 37 °C. The resulting Spinach RNA aptamers specifically bind to fluorogens such as 3,5-difluoro-4-hydroxybenzylidene imidazolinone and emit a highly enhanced fluorescence signal, which is clearly distinguished from the signal emitted in the absence of the target nucleic acid. With the proposed strategy, concentrations of target nucleic acids selected from the genome of Salmonellaenterica serovar Typhi (S. Typhi) were quantitatively determined with high selectivity. In addition, the practical applicability of the method was demonstrated by performing spike-and-recovery experiments with S. Typhi in human serum.

The impact of organic extracts of seasonal PM2.5 on primary human lung epithelial cells and their chemical characterization.

Lung epithelial cells serve as the first line of defense against various inhaled pollutant particles. To investigate the adverse health effects of organic components of fine particulate matter (PM2.5) collected in Seoul, South Korea, we selected 12 PM2.5 samples from May 2016 to January 2017 and evaluated the effects of organic compounds of PM2.5 on inflammation, cellular aging, and macroautophagy in human lung epithelial cells isolated directly from healthy donors. Organic extracts of PM2.5 specifically induced neutrophilic chemokine and interleukin-8 expression via extracellular signal-regulated kinase activation. Moreover, PM2.5 significantly increased the expression of aging markers (p16, p21, and p27) and activated macroautophagy. Average mass concentrations of organic and elemental carbon had no significant correlations with PM2.5 effects. However, polycyclic aromatic hydrocarbons and n-alkanes were the most relevant components of PM2.5 that correlated with neutrophilic inflammation. Vegetative detritus and residential bituminous coal combustion sources strongly correlated with neutrophilic inflammation, aging, and macroautophagy activation. These data suggest that the chemical composition of PM2.5 is important for determining the adverse health effects of PM2.5. Our study provides encouraging evidence to regulate the harmful components of PM2.5 in Seoul.

Cigarette smoke extract-induced downregulation of p300 is responsible for the impaired inflammatory cytokine response of macrophages.

Patients with chronic obstructive pulmonary disease (COPD) are susceptible to infection owing to the impaired immune function of alveolar macrophages. This is presumed to be caused, at least partially, by cigarette smoke (CS), which is a major risk factor for COPD. Although CS has been reported to inhibit Toll-like receptor (TLR) function and phagocytosis in macrophages, the molecular mechanism of CS-mediated impairment of macrophage immune function has not been completely elucidated. We investigated the effects of CS extracts (CSE) on macrophage immune function and its molecular mechanism. We assessed lipopolysaccharide (LPS, TLR4 ligand)-, Pam3CSK4 (TLR2 ligand)-, or CpG-oligodeoxynucleotide (TLR9 ligand)-induced IL-6, TNF-α, and IL-1ß production in macrophages. Upregulation of IL-6, TNF-α, and IL-1ß mRNA and protein by TLR ligands was suppressed on treatment with CSE. However, LPS-induced MAP kinase activation, IκBα degradation, and nuclear translocation of NF-κB were not impeded by CSE. In contrast, CSE significantly suppressed NF-κB transcriptional activity in the nucleus. We found that p300, which acetylates RelA/p65 at lysine 310, and acetyl-p65 (K310) were downregulated upon CSE treatment. Knock-down of p300 suppressed LPS-induced acetylation of NF-κB p65 and production of inflammatory cytokine. To summarize, these results suggest that CSE impair cytokine response by decreasing the expression levels of p300.

Cereblon contributes to the development of pulmonary fibrosis via inactivation of adenosine monophosphate-activated protein kinase α1.

Pulmonary fibrosis is a progressive and lethal lung disease characterized by the proliferation and differentiation of lung fibroblasts and the accumulation of extracellular matrices. Since pulmonary fibrosis was reported to be associated with adenosine monophosphate-activated protein kinase (AMPK) activation, which is negatively regulated by cereblon (CRBN), we aimed to determine whether CRBN is involved in the development of pulmonary fibrosis. Therefore, we evaluated the role of CRBN in bleomycin (BLM)-induced pulmonary fibrosis in mice and in transforming growth factor-beta 1 (TGF-ß1)-induced differentiation of human lung fibroblasts. BLM-induced fibrosis and the mRNA expression of collagen and fibronectin were increased in the lung tissues of wild-type (WT) mice; however, they were significantly suppressed in Crbn knockout (KO) mice. While the concentrations of TGF-ß1/2 in bronchoalveolar lavage fluid were increased via BLM treatment, they were similar between BLM-treated WT and Crbn KO mice. Knockdown of CRBN suppressed TGF-ß1-induced activation of small mothers against decapentaplegic 3 (SMAD3), and overexpression of CRBN increased it. TGF-ß1-induced activation of SMAD3 increased α-smooth muscle actin (α-SMA) and collagen levels. CRBN was found to be colocalized with AMPKα1 in lung fibroblasts. CRBN overexpression inactivated AMPKα1. When cells were treated with metformin (an AMPK activator), the CRBN-induced activation of SMAD3 and upregulation of α-SMA and collagen expression were significantly suppressed, suggesting that increased TGF-ß1-induced activation of SMAD3 via CRBN overexpression is associated with AMPKα1 inactivation. Taken together, these data suggest that CRBN is a profibrotic regulator and maybe a potential target for treating lung fibrosis.

Synergistic cycles of protease activity and inflammation via PPARγ degradation in chronic obstructive pulmonary disease.

Inflammation, oxidative stress, and protease-antiprotease imbalance have been suggested to be a pathogenic triad in chronic obstructive pulmonary disease (COPD). However, it is not clear how proteases interact with components of inflammatory pathways. Therefore, this study aimed to evaluate the effect of neutrophil elastase (NE) on lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) production and determine the molecular mechanism in human bronchial epithelial cells (HBECs). Immortalized bronchial epithelial cells and primary HBECs were used to investigate the impact of NE on LPS-induced IL-8 production. The molecular mechanism by which NE modulated LPS-induced IL-8 production was confirmed in elastase-treated C57BL/6 mice and primary HBECs obtained from COPD patients and healthy controls. The results showed that NE treatment synergistically augmented LPS-induced IL-8 production in both immortalized bronchial epithelial cells and primary HBECs. NE partially degraded peroxisome proliferator-activated receptor gamma (PPARγ), which is known to regulate IL-8 production in the nucleus. Treatment with a PPARγ agonist and overexpression of PPARγ reversed the NE-induced synergistic increase in LPS-induced IL-8 production. Moreover, PPARγ levels were lower in lung homogenates and lung epithelial cells from elastase-treated mice than in those from saline-treated mice. In accordance with the findings in mice, PPARγ levels were lower in primary HBECs from COPD patients than in those from healthy never-smokers or healthy smokers. In conclusion, a vicious cycle of mutual augmentation of protease activity and inflammation resulting from PPARγ degradation plays a role in the pathogenesis of COPD.

Promoter engineering improves transcription efficiency in biomolecular assays.

We identified a novel 12 bp promoter that significantly increased transcription efficiency. Unlike the standard 20 bp promoter, which contains both recognition and initiation regions, the new promoter contains only a recognition region and is more suitable for diagnostic applications due to its smaller size. This promoter effectively produced different light-up RNA aptamers via transcription. Moreover, we used the promoter to analyze RNase H activity and achieved a detection limit of 0.009 U mL-1, which was significantly better than that achieved via previous methods. We propose that the new promoter may serve as a key component in various diagnostic applications.

Cigarette smoke extract decreased basal and lipopolysaccharide-induced expression of MARCO via degradation of p300.

BACKGROUND AND OBJECTIVE: Alveolar macrophages of patients with COPD display impaired cytokine release and diminished phagocytosis. COPD exacerbations exhibit immune dysfunction towards the respiratory pathogens. CS and CSE were reported to aggravate bacterial infections in COPD patients. METHODS: MARCO is highly expressed in lungs and is involved in pathogen clearance. We investigated the effect of CSE on MARCO expression and its regulatory mechanisms. After relevant siRNA transfection and treatment with CSE and/or LPS, we measured the levels of MARCO by q-RT PCR, immunoblotting and flow cytometry. Immunofluorescence staining and immunoprecipitation were used to evaluate the mechanism. RESULTS: CSE decreased LPS-induced expression of MARCO mRNA and protein. Upregulation of MARCO by LPS was Nrf2-dependent. Nrf2 knockdown significantly suppressed LPS-induced increase in MARCO transcripts. CSE did not block nuclear translocation of Nrf2 in LPS-treated cells, but rather CSE itself strongly accumulated Nrf2 in the nucleus through the degradation of its cytoplasmic inhibitor, KEAP1. However, CSE markedly suppressed LPS-induced Nrf2 acetylation. Histone acetyltransferase p300/CBP directly acetylates Nrf2, which augments promoter-specific DNA binding of Nrf2. Our results reveal CSE-induced polyubiquitinylation and subsequent degradation of p300 via the proteasome. Pretreatment with proteasome inhibitors completely blocked CSE-induced degradation of p300 and suppression of MARCO expression. CONCLUSION: These findings suggest that CSE decreases MARCO expression via the proteasomal degradation of p300 in macrophages, which may be in part responsible for impaired bacterial phagocytosis.

Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells.

IκB, a cytoplasmic inhibitor of nuclear factor-κB (NF-κB), is reportedly degraded via the proteasome. However, we recently found that long-term incubation with proteasome inhibitors (PIs) such as PS-341 or MG132 induces IκBα degradation via an alternative pathway, lysosome, which results in NF-κB activation and confers resistance to PI-induced lung cancer cell death. To enhance the anti-cancer efficacy of PIs, elucidation of the regulatory mechanism of PI-induced IκBα degradation is necessary. Here, we demonstrated that PI upregulates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) via both de novo protein synthesis and Kelch-like ECH-associated protein 1 (KEAP1) degradation, which is responsible for IκBα degradation via macroautophagy activation. PIs increased the protein level of light chain 3B (LC3B, macroautophagy marker), but not lysosome-associated membrane protein 2a (Lamp2a, the receptor for chaperone-mediated autophagy) in NCI-H157 and A549 lung cancer cells. Pretreatment with macroautophagy inhibitor or knock-down of LC3B blocked PI-induced IκBα degradation. PIs up-regulated Nrf2 by increasing its transcription and mediating degradation of KEAP1 (cytoplasmic inhibitor of Nrf2). Overexpression of dominant-negative Nrf2, which lacks an N-terminal transactivating domain, or knock-down of Nrf2 suppressed PI-induced LC3B protein expression and subsequent IκBα degradation. Thus, blocking of the Nrf2 pathway enhanced PI-induced cell death. These findings suggest that Nrf2-driven induction of LC3B plays an essential role in PI-induced activation of the IκB/NF-κB pathway, which attenuates the anti-tumor efficacy of PIs.

Globular Adiponectin Exerts a Pro-Inflammatory Effect via IκB/NF-κB Pathway Activation and Anti-Inflammatory Effect by IRAK-1 Downregulation.

Adiponectin, a hormone produced by adipose tissue, is very abundant in plasma, and its anti- and pro-inflammatory effects are reported. However, the mechanisms of these pro- and anti-inflammatory effects are not fully defined. Herein, we evaluated the dual inflammatory response mechanism of adiponectin in macrophages. Short-term globular adiponectin (gAd) treatment induced IκBα degradation, NF-κB nuclear translocation, and TNF-α production in RAW 264.7 cells. Polymyxin B pretreatment did not block gAd-induced IκBα degradation, and heated gAd was unable to degrade IκBα, suggesting that the effects of gAd were not due to endotoxin contamination. gAd activated IKK and Akt, and inhibition of either IKK or Akt by dominant-negative IKKß (DN-IKKß) or DN-Akt overexpression blocked gAd-induced IκBα degradation, suggesting that short-term incubation with gAd mediates inflammatory responses by activating the IκB/NF-κB and PI3K/Akt pathways. Contrastingly, long-term stimulation with gAd induced, upon subsequent stimulation, tolerance to gAd, lipopolysaccharide, and CpG-oligodeoxynucleotide, which is associated with gAd-induced downregulation of IL-receptor-associated kinase-1 (IRAK-1) due to IRAK-1 transcriptional repression. Conclusively, our findings demonstrate that the pro- and anti-inflammatory responses to gAd in innate immune cells are time-dependent, and mediated by the activation of the IκB/NF-κB pathway, and IRAK-1 downregulation, respectively.

IL-6-mediated cross-talk between human preadipocytes and ductal carcinoma in situ in breast cancer progression.

BACKGROUND: The function of preadipocytes in the progression of early stage breast cancer has not been fully elucidated at the molecular level. To delineate the role of preadipocytes in breast cancer progression, we investigated the cross-talk between human breast ductal carcinoma in situ (DCIS) cells and preadipocytes with both an in vitro culture and xenograft tumor model. METHODS: GFP or RFP was transduced into human DCIS cell line MCF10DCIS.com cells or preadipocytes using lentivirus. Cell sorter was used to separate pure, viable populations of GFP- or RFP-transduced cells. Cell viability and proliferation was assessed by crystal violet assays and cell migration and invasion capability was assayed by the transwell strategy. Gene and protein levels were measured by western blot, RT-PCR and immunostaining. Adipokines and cytokines were quantified using ELISA. Human tumor xenografts in a nude mice model were used. Ultrasound imaging of tumors was performed to evaluate the therapeutic potential of a IL-6 neutralizing antibody. RESULTS: In the co-culture system with the MCF10DCIS.com and preadipocytes, MCF10DCIS.com proliferation, migration and invasion were enhanced by preadipocytes. Preadipocytes exhibited in an increased IL-6 secretion and cancer-associated fibroblast markers expression, FSP1 and α-SMC in co-culture with MCF10DCIS.com or in MCF10DCIS.com conditioned media, whereas the adipocyte differentiation capacity was suppressed by co-culture with MCF10DCIS.com. A neutralizing antibody of IL-6 or IL-6R suppressed the promotion of MCF10DCIS.com proliferation and migration by co-culture with preadipocytes. In the xenograft tumor model, the tumor growth of MCF10DCIS.com was enhanced by the co-injection of preadipocytes, and the administration of IL-6 neutralizing antibodies resulted in potent effects on tumor inhibition. CONCLUSIONS: Our findings suggest that IL-6-mediated cross-talk between preadipocytes and breast DCIS cells can promote the progression of early stage breast cancer. Therefore, blocking IL-6 signaling might be a potential therapeutic strategy for breast DCIS characterized by pathological IL-6 overproduction.

Cigarette smoke extract enhances neutrophil elastase-induced IL-8 production via proteinase-activated receptor-2 upregulation in human bronchial epithelial cells.

Although inflammation, oxidative stress, and protease-antiprotease imbalance have been referred to as a pathogenic triad in chronic obstructive pulmonary disease (COPD), little is known about how they interact. The objectives of this study were to elucidate the effect of cigarette smoke extract (CSE) on the neutrophil elastase (NE)-induced inflammatory response and its molecular mechanism in bronchial epithelial cells. We observed that NE activated extracellular signal-regulated kinase (ERK) and induced IL-8 production. Blocking ERK activation using a MEK inhibitor (U0126) suppressed NE-induced IL-8 secretion and knockdown of proteinase-activated receptor 2 (PAR2) using siRNAs inhibited both NE-induced ERK activation and subsequent IL-8 release, suggesting that NE-induced IL-8 production is dependent on PAR2-mediated ERK activation. Interestingly, pre-exposure to CSE markedly enhanced NE-induced IL-8 production. As PAR2 acts as a receptor for NE, we next investigated the effect of CSE on PAR2 expression as a molecular mechanism for the increased IL-8 production induced by NE in CSE exposed cells. CSE, but not NE, increased the expression of PAR2 mRNA and surface membrane protein. Inhibition of p38 MAPK reduced PAR2 expression induced by CSE while inhibition of the ERK and Akt pathway had no effect. Consequently, p38 inhibition significantly abrogated CSE-induced enhancement of IL-8 production in NE-treated cells. Of note, we observed increased PAR2 levels in lung homogenates and lung epithelial cells from CSE-treated mice and from both smokers and patients with COPD. Taken together, these results suggest that CSE upregulates PAR2 in normal human bronchial epithelial cells, thereby enhancing the inflammatory response to NE.