MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1−/− mice with Muc1+/+ littermates following intranasal instillation of PA or flagellin. Compared with Muc1+/+ mice, Muc1−/− mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-α and KC in bronchoalveolar lavage fluid, higher levels of TNF-α in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.

In this study, we investigated the regulation and mechanism of IL-8 expression by A549 human lung carcinoma cells treated with neutrophil elastase (NE). NE-treated cells exhibited significantly higher IL-8 protein levels in culture media compared with cells treated with vehicle alone. Blocking of gene transcription with actinomycin D suggested that NE stimulated IL-8 synthesis via increased mRNA expression, which was verified by real-time RT-PCR. NE activated the IL-8 promoter but did not alter the stability of its mRNA, confirming that the protease induced IL-8 synthesis through increased gene transcription. The results from the use of chemical inhibitors and mutant gene constructs against various signal transduction components seem to suggest the linear signaling pathway involving the activation of PKC-delta --> dual oxidase 1 --> reactive oxygen species --> TNF-alpha-converting enzyme --> EGF receptor --> p38 --> NF-kappaB for NE-activated IL-8 gene expression. A NF-kappaB potential binding site, located between nucleotides -82 and -69 of the IL-8 promoter, was identified as necessary for NE-induced IL-8 transcription. We conclude that NE increases IL-8 transcription through p38/NF-kappaB activation via EGFR transactivation.

The current study was conducted to elucidate the mechanism through which TNF-alpha stimulates expression of MUC1, a membrane-tethered mucin. A549 human lung alveolar cells treated with TNF-alpha exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Increased MUC1 protein levels were correlated with significantly higher levels of MUC1 mRNA in TNF-alpha-treated cells compared with controls. However, TNF-alpha did not alter MUC1 transcript stability, implying increased de novo transcription induced by the cytokine. TNF-alpha increased MUC1 gene promoter activity in A549 cells transfected with a promoter-luciferase reporter plasmid. Both U0126, an inhibitor of MEK1/2, and dominant negative ERK1 prevented TNF-alpha-induced MUC1 promoter activation, and anti-TNFR1 antibody blocked TNF-alpha-stimulated ERK1/2 activation. MUC1 promoter activation by TNF-alpha also was blocked by mithramycin A, an inhibitor of Sp1, as well as either deletion or mutation of a putative Sp1 binding site in the MUC1 promoter located between nucleotides -99 and -90. TNF-alpha-stimulated binding of Sp1 to the MUC1 promoter in intact cells was demonstrated by chromatin immunoprecipitation assay. We conclude that TNF-alpha induces MUC1 gene transcription through a TNFR1 --> MEK1/2 --> ERK1 --> Sp1 pathway.

Background— The acetylcholine-activated K + current ( I K,ACh ) is a novel candidate for atrial-specific antiarrhythmic therapy. The present study investigates the involvement of I K,ACh in atrial fibrillation (AF) using NTC-801, a novel potent and selective I K,ACh blocker. Methods and Results— The effects of NTC-801, substituted 4-(aralkylamino)-2,2-dimethyl-3,4-dihydro- 2H -benzopyran-3-ol, on I K,ACh and other cardiac ionic currents ( I Na , I CaL , I to , I Kur , I Kr , I Ks , I Kl , I KATP , and I f ) and on atrial and ventricular action potentials were examined in vitro. NTC-801 potently inhibited carbachol-induced I K,ACh in guinea pig atrial cells and the GIRK1/4 current in Xenopus oocytes with IC 50 values of 5.7 and 0.70 nmol/L, respectively. NTC-801 selectively inhibited I K,ACh >1000-fold over other cardiac ionic currents. NTC-801 (10 to 100 nmol/L) reversed the action potential duration (APD 90 ) shortened by carbachol or adenosine in atrial cells, whereas it did not affect APD 90 at 100 nmol/L in ventricular cells. Antiarrhythmic effects of NTC-801 were evaluated in 3 AF models in vivo. NTC-801 significantly prolonged atrial effective refractory period without affecting ventricular effective refractory period under vagal nerve stimulation. NTC-801 dose-dependently converted AF to normal sinus rhythm in both vagal nerve stimulation–induced (0.3 to 3 μg · kg −1 · min −1 IV) and aconitine-induced (0.01 to 0.1 mg/kg IV) models. In a rapid atrial pacing model, NTC-801 (3 μg · kg −1 · min −1 IV) significantly decreased AF inducibility with a prolonged atrial effective refractory period that was frequency-independent. Conclusions— A selective I K,ACh blockade induced by NTC-801 exerted anti-AF effects mediated by atrial-selective effective refractory period prolongation. These findings suggest that I K,ACh may be important in the development and maintenance of AF.