title: An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis.
abstract: BACKGROUND
  Subcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists.
  
  
  METHODS
  FLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy.
  
  
  RESULTS
  5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca(2+)-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB4. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP.
  
  
  CONCLUSIONS
  The cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging.
  
  
  CONCLUSIONS
  We present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.
authors.affiliation: Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Gerstmeier
authors.affiliation: Institute of Transfusion Medicine, Jena University Hospital, 07743 Jena, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Weinigel
authors.affiliation: Institute of Transfusion Medicine, Jena University Hospital, 07743 Jena, Germany.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Barz
authors.affiliation: Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Werz
authors.affiliation: Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Garscha
mesh_terms: D058927:5-Lipoxygenase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D061207:Calcium Ionophores / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D015289:Leukotrienes / Q000096:biosynthesis* / Q000235:genetics; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology*; D002722:Chlorobenzenes
chemical_list: D058927:5-Lipoxygenase-Activating Proteins; D001094:Arachidonate 5-Lipoxygenase; D000001:Calcimycin; D061207:Calcium Ionophores; D007211:Indoles; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; C548592:ALOX5AP protein, human; C060893:MK-886; D002722:Chlorobenzenes
keywords: 12-Lipoxygenase; 5-Lipoxygenase; 5-Lipoxygenase-activating protein; Arachidonic acid; HEK293 cells; MK886