title: 5-Hydroxy-7-methoxyflavone inhibits N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced superoxide anion production by specific modulate membrane localization of Tec with a PI3K independent mechanism in human neutrophils.
abstract: Respiratory burst mediates crucial bactericidal mechanism in neutrophils. However, undesirable respiratory burst leads to pathological inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 5-hydroxy-7-methoxyflavone (MCL-1), a lignan extracted from the leaves of Muntingia calabura L. (Tiliaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G release in human neutrophils. Signaling pathways regulated by MCL-1 to oppose fMLP-induced respiratory burst were evaluated by membrane localization of Tec induced by fMLP and by immunoblotting analysis of downstream phosphorylation targets of Tec. Briefly, MCL-1 specific inhibited fMLP-induced superoxide anion production in a concentration-dependent (IC(50)=0.16±0.01 μM) and Tec kinase-dependent manner, however, MCL-1 did not affect fMLP-induced cathepsin G release. Further, MCL-1 suppressed fMLP-induced Tec translocation from the cytosol to the inner leaflet of the plasma membrane, and subsequently activation of phospholipase Cγ2 (PLCγ2). Moreover, MCL-1 attenuated PLCγ2 activity and intracellular calcium concentration notably through extracellular calcium influx. Consequently, fMLP-induced phosphorylation of protein kinase C (PKC) and membrane localization of p47(phox) were decreased by MCL-1 in a Tec-dependent manner, while the phosphorylation of extracellular signal-regulated kinase (ERK), p38, AKT and Src tyrosine kinase family remained unaffected. In addition, MCL-1 neither inhibited NADPH oxidase activity nor increased cyclicAMP levels. MCL-1 specific opposes fMLP-mediated respiratory burst by inhibition of membrane localization of Tec and subsequently interfered with the activation of PLCγ2, protein kinase C, and p47(phox).
authors.affiliation: Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan. liaoch@mail.cgu.edu.tw
authors.forename: Hsiang-Ruei
authors.initials: HR
authors.lastname: Liao
authors.forename: Jih-Jung
authors.initials: JJ
authors.lastname: Chen
authors.forename: Yin-Huan
authors.initials: YH
authors.lastname: Chien
authors.forename: Shinn-Zhi
authors.initials: SZ
authors.lastname: Lin
authors.forename: Shunchih
authors.initials: S
authors.lastname: Lin
authors.forename: Ching-Ping
authors.initials: CP
authors.lastname: Tseng
mesh_terms: D000328:Adult; D002118:Calcium / Q000378:metabolism; D056649:Cathepsin G / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D029646:Tiliaceae / Q000737:chemistry
chemical_list: C574159:5-hydroxy-7-methoxyflavone; D005419:Flavonoids; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000242:Cyclic AMP; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; D051966:Phospholipase C gamma; C534094:CTSG protein, human; D056649:Cathepsin G; D002118:Calcium