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Abstract: Oxidized cholesterols belong to a subgroup of oxLDLs which play major roles in atherosclerosis. In order to investigate the contribution of oxysterols from oxLDLs in atherosclerosis, cholesterol-3-beta, 5-alpha, 6-beta-triol (α-Triol) was studied in human umbilical vein endothelial cells. We found that α-Triol concentration- and time-dependently enhanced COX-2 protein expression and mRNA production followed by PGE2 generation in human umbilical vein endothelial cells. In addition, α-Triol upregulated peNOS1177 protein phosphorylation and concentration-dependently increased nitric oxide production. eNOS1177 phosphorylation was abrogated by the PI3K inhibitor, LY294002. In studying the mechanisms involved in α-Triol-induced COX-2/PGE2 production, inhibitors of NOS, PI3K, p38, and NF-κB, effectively attenuated COX-2 protein induction and mRNA expression, suggesting that the PI3K-Akt-eNOS pathway, p38MAPK, and NF-κB are involved in α-Triol-induced COX-2 expression, and following increases in p38 and Akt phosphorylation, the concentration-dependent inhibition of COX-2 protein expression by L-NAME further suggested their involvement at the translation level. We concluded that α-Triol increases COX-2 mRNA and protein expression via coordination with the PI3K-Akt-eNOS pathway and NF-κB. Moreover, COX-2 gene expression might be regulated by activated p38 MAPK in another unknown regulation pathway. Our findings also suggested that α-Triol might contribute to the effect of induced atherosclerosis in humans through COX-2 production in endothelial cells. [Copyright &y& Elsevier]

The transcription factor PsrA regulates fatty acid metabolism, the type III secretion system, and quinolone signaling quorum sensing system in Pseudomonas aeruginosa . To explore additional roles of PsrA in P. aeruginosa , this study engineered a P. aeruginosa PAO1 strain to carry a recombinant plasmid with the psrA gene (pMMB psrA ) and examined the impact of elevated psrA expression to the bacterium. Transcriptomic analysis revealed that PsrA significantly downregulated genes encoding the master quorum-sensing regulators, RhlR and LasR, and influenced many quorum-sensing-associated genes. The role of PsrA in quorum sensing was further corroborated by testing autoinducer synthesis in PAO1 [pMMB psrA ] using two reporter bacteria strains Chromobacterium violaceum CV026 and Escherichia coli [pSB1075], which respond to short- and long-chain acyl homoserine lactones, respectively. Phenotypic comparisons of isogenic Δ psrA , Δ lasR , and Δ psrA Δ lasR mutants revealed that the reduced elastase, caseinase, and swarming activity in PAO1 [pMMB psrA ] were likely mediated through LasR. Additionally, electrophoretic mobility shift assays demonstrated that recombinant PsrA could bind to the lasR promoter at a 5'-AAACGTTTGCTT-3' sequence, which displays moderate similarity to the previously reported consensus PsrA binding motif. Furthermore, the PsrA effector molecule oleic acid inhibited PsrA binding to the lasR promoter and restored several quorum sensing-related phenotypes to wild-type levels. These findings suggest that PsrA regulates certain quorum-sensing phenotypes by negatively regulating lasR expression, with oleic acid acting as a crucial signaling molecule., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kok, Tsai, Liao, Lo, Cheng, Lin and Chang.)