title: Macrophage-activating lipopeptide-2 requires Mal and PI3K for efficient induction of heme oxygenase-1.
abstract: OBJECTIVE
  This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes.
  
  
  METHODS
  Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay.
  
  
  RESULTS
  MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, LY294002.
  
  
  CONCLUSIONS
  These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Xiaoxing
authors: X
authors: You
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Liangzhuan
authors: L
authors: Liu
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Yanhua
authors: Y
authors: Zeng
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Ranhui
authors: R
authors: Li
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China; Department of Clinical Laboratory, The Affiliated Nanhua Hospital of University of South China, Hengyang City, P.R. China.
authors: Jun
authors: J
authors: He
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China; Department of Clinical Laboratory, Changsha Central Hospital, Chagnsha City, P.R. China.
authors: Xiaohua
authors: X
authors: Ma
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Chuanhao
authors: C
authors: Jiang
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Cuiming
authors: C
authors: Zhu
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Liesong
authors: L
authors: Chen
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Minjun
authors: M
authors: Yu
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Guangli
authors: G
authors: Ou
authors: Institution of Pathogenic Biology, Medical College, University of South China, Hengyang City, P.R. China.
authors: Yimou
authors: Y
authors: Wu
mesh_terms: D002460:Cell Line; D004790:Enzyme Induction; D051547:Heme Oxygenase-1 / Q000096:biosynthesis*; D006801:Humans; D055666:Lipopeptides / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051193:Toll-Like Receptors / Q000502:physiology
chemical_list: D055666:Lipopeptides; D051193:Toll-Like Receptors; C106335:macrophage stimulatory lipopeptide 2; D051547:Heme Oxygenase-1; D019869:Phosphatidylinositol 3-Kinases