Wnt family members play diverse roles in development and disease. Noncanonical Wnt ligands can inhibit canonical Wnt signaling depending on the cellular context; however, the underlying mechanism of this antagonism remains poorly understood. Here we identify a specific mechanism of orphan nuclear receptor RORalpha-mediated inhibition of canonical Wnt signaling in colon cancer. Wnt5a/PKCalpha-dependent phosphorylation on serine residue 35 of RORalpha is crucial to link RORalpha to Wnt/beta-catenin signaling, which exerts inhibitory function of the expression of Wnt/beta-catenin target genes. Intriguingly, there is a significant correlation of reduction of RORalpha phosphorylation in colorectal tumor cases compared to their normal counterpart, providing the clinical relevance of the findings. Our data provide evidence for a role of RORalpha, functioning at the crossroads between the canonical and the noncanonical Wnt signaling pathways, in mediating transrepression of the Wnt/beta-catenin target genes, thereby providing new approaches for the development of therapeutic agents for human cancers.

c Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na ؉ dependent. Consistent with the finding of a Na ؉ -dependent Rnf complex is the presence of a conserved Na ؉ -binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H 2 ؉ CO 2 . The energetic benefit may be one reason that butyrate is formed only from CO but not from H 2 ؉ CO 2 .A cetogenic bacteria are a phylogenetically diverse group of strictly anaerobic bacteria able to reduce two molecules of CO 2 to acetate by the Wood-Ljungdahl pathway (WLP) (1-4). Electrons may derive from molecular hydrogen (autotrophic growth), from carbon monoxide, or from organic donors (heterotrophic growth) such as hexoses, pentoses, formate, lactate, alcohols, or methyl group donors (1). Not only does the WLP provide the cell with organic material for biomass formation, but it is also coupled to energy conservation for ATP supply by a chemiosmotic mechanism (2, 5). Every acetogen examined to date uses reduced ferredoxin (Fd) as the electron donor for an ion-translocating membrane protein complex, and acetogens can have either an Fd:NAD ϩ oxidoreductase (Rnf) or an Fd:H ϩ oxidoreductase (Ech) complex for generation of an ion motive force (5). In both cases, the ion gradient can be either an H ϩ or an Na ϩ gradient. The electrochemical ion gradient thus established is then used by a membrane bound, H ϩ -or Na ϩ -translocating F 1 F o ATP synthase (2).Acetate production from CO 2 proceeds via formate that is converted to formyl-tetrahydrofolate (THF) in an ATP-consuming reaction (6). Water is split off from formyl-THF to yield methenyl-THF, which is reduced via methylene-THF to methyl-THF. The latter is condensed with CO (derived from another molecule of CO 2 ) and coenzyme A (CoA) to acetyl-CoA, which is the starting molecule for biosynthetic reactions (4,7,8). Acetyl-CoA is also the precursor of the end product, acetate, that is produced by the enzymes acetyltransferase and acetate kinase. ATP production in the acetate kinase reaction is of special...

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Background: Interaction between oxidative stress (CSE) and protease (NE) may contribute to COPD pathogenesis. Results: Although NE enhances CSE-induced IL-8, it suppresses VEGF production, which is due to degradation by uptake of NE into bronchial epithelial cells. Conclusion: NE contributes to the pathogenesis of COPD by enhancing inflammation and apoptosis.Significance: This provides a molecular mechanism for understanding COPD pathogenesis.

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.