Decreased expression of the NF-κB family member RelB in lung fibroblasts from Smokers with and without COPD potentiates cigarette smoke-induced COX-2 expression.

BACKGROUND: Heightened inflammation, including expression of COX-2, is associated with COPD pathogenesis. RelB is an NF-κB family member that attenuates COX-2 in response to cigarette smoke by a mechanism that may involve the miRNA miR-146a. There is no information on the expression of RelB in COPD or if RelB prevents COX-2 expression through miR-146a. METHODS: RelB, Cox-2 and miR-146a levels were evaluated in lung fibroblasts and blood samples derived from non-smokers (Normal) and smokers (At Risk) with and without COPD by qRT-PCR. RelB and COX-2 protein levels were evaluated by western blot. Human lung fibroblasts from Normal subjects and smokers with and without COPD, along with RelB knock-down (siRNA) in Normal cells, were exposed to cigarette smoke extract (CSE) in vitro and COX-2 mRNA/protein and miR-146a levels assessed. RESULTS: Basal expression of RelB mRNA and protein were significantly lower in lung cells derived from smokers with and without COPD, the latter of which expressed more Cox-2 mRNA and protein in response to CSE. Knock-down of RelB in Normal fibroblasts increased Cox-2 mRNA and protein induction by CSE. Basal miR-146a levels were not different between the three groups, and only Normal fibroblasts increased miR-146a expression in response to smoke. There was a positive correlation between systemic RelB and Cox-2 mRNA levels and circulating miR-146a levels were higher only in GOLD stage I subjects. CONCLUSIONS: Our data indicate that RelB attenuates COX-2 expression in lung structural cells, such that loss of pulmonary RelB may be an important determinant in the aberrant, heightened inflammation associated with COPD pathogenesis.

Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability.

Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.

The aryl hydrocarbon receptor suppresses cigarette-smoke-induced oxidative stress in association with dioxin response element (DRE)-independent regulation of sulfiredoxin 1.

The aryl hydrocarbon receptor (AhR) is a ubiquitously expressed receptor/transcription factor that mediates toxicological responses of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Emerging evidence indicates that the AhR suppresses apoptosis and proliferation independent of exogenous ligands, including suppression of apoptosis by cigarette smoke, a key risk factor for chronic obstructive pulmonary disease (COPD). As cigarette smoke is a potent inducer of oxidative stress, a feature that may contribute to the development of COPD, we hypothesized that the AhR prevents smoke-induced apoptosis by regulating oxidative stress. Utilizing primary lung fibroblasts derived from AhR(+/+) and AhR(-/-) mice as well as A549 human lung adenocarcinoma cells deficient in AhR expression (A549-AhR(ko)), we first show that AhR(-/-) fibroblasts and A549-AhR(ko) epithelial cells have a significant increase in cigarette smoke extract (CSE)-induced oxidative stress compared to wild type. CSE induced a significant increase in the mRNA expression of key antioxidant genes, including Nqo1 and Srxn1, predominantly in AhR(+/+) fibroblasts, with significantly less induction in AhR(-/-) cells. The induction of Srxn1, but not Nqo1, was independent of dioxin-response element (DRE) binding as AhR(DBD/DBD) lung fibroblasts, which express an AhR incapable of binding the DRE, increased Srxn1 to a degree similar to wild-type cells in response to CSE. There was no difference in Nrf2 expression or activation based on AhR expression. Lung fibroblasts derived from COPD subjects have significantly less AhR protein expression compared with both never-smokers (Normal) and smokers (At Risk). Consequently, COPD-derived fibroblasts were less robust in their induction of both Nqo1 and Srxn1 mRNA after exposure to CSE, which also failed to activate the AhR in the COPD fibroblasts. Taken together, these results support a new role for the AhR in regulating antioxidant defense in lung structural cells, such that low AhR expression may facilitate the development or progression of COPD.

Aryl hydrocarbon receptor-dependent retention of nuclear HuR suppresses cigarette smoke-induced cyclooxygenase-2 expression independent of DNA-binding.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to man-made environmental toxicants, has emerged as an endogenous regulator of cyclooxygenase-2 (Cox-2) by a mechanism that is poorly understood. In this study, we first used AhR-deficient (AhR(-/-) ) primary pulmonary cells, together with pharmacological tools to inhibit new RNA synthesis, to show that the AhR is a prominent factor in the destabilization of Cox-2 mRNA. The destabilization of Cox-2 mRNA and subsequent suppression of cigarette smoke-induced COX-2 protein expression by the AhR was independent of its ability to bind the dioxin response element (DRE), thereby differentiating the DRE-driven toxicological AhR pathway from its anti-inflammatory abilities. We further describe that the AhR destabilizes Cox-2 mRNA by sequestering HuR within the nucleus. The role of HuR in AhR stabilization of Cox-2 mRNA was confirmed by knockdown of HuR, which resulted in rapid Cox-2 mRNA degradation. Finally, in the lungs of AhR(-/-) mice exposed to cigarette smoke, there was little Cox-2 mRNA despite robust COX-2 protein expression, a finding that correlates with almost exclusive cytoplasmic HuR within the lungs of AhR(-/-) mice. Therefore, we propose that the AhR plays an important role in suppressing the expression of inflammatory proteins, a function that extends beyond the ability of the AhR to respond to man-made toxicants. These findings open the possibility that a DRE-independent AhR pathway may be exploited therapeutically as an anti-inflammatory target.

The Pseudomonas aeruginosa autoinducer 3O-C12 homoserine lactone provokes hyperinflammatory responses from cystic fibrosis airway epithelial cells.

The discovery of novel antiinflammatory targets to treat inflammation in the cystic fibrosis (CF) lung stands to benefit patient populations suffering with this disease. The Pseudomonas aeruginosa quorum sensing autoinducer N-3-oxododecanoyl homoserine lactone (3O-C12) is an important bacterial virulence factor that has been reported to induce proinflammatory cytokine production from a variety of cell types. The goal of this study was to examine the ability of 3O-C12 to induce proinflammatory cytokine production in normal and CF bronchial epithelial cells, and better understand the cellular mechanisms by which this cytokine induction occurs. 3O-C12 was found to induce higher levels of IL-6 production in the CF cell lines IB3-1 and CuFi, compared to their corresponding control cell lines C38 and NuLi. Systems biology and network analysis revealed a high predominance of over-represented innate immune pathways bridged together by calcium-dependant transcription factors governing the transcriptional responses of A549 airway cells to stimulation with 3O-C12. Using calcium-flux assays, 3O-C12 was found to induce larger and more sustained increases in intracellular calcium in IB3-1 cells compared to C38, and blocking this calcium flux with BAPTA-AM reduced the production of IL-6 by IB3-1 to the levels produced by C38. These data suggest that 3O-C12 induces proinflammatory cytokine production in airway epithelial cells in a calcium-dependent manner, and that dysregulated calcium storage or signalling in CF cells results in an increased production of proinflammatory cytokines.