An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

• Published in: Biochimica et biophysica acta Vol. 1840; no. 9; pp. 2961 - 2969
• Main Authors: Gerstmeier, Jana, Weinigel, Christina, Barz, Dagmar, Werz, Oliver, Garscha, Ulrike
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2014
• Subjects: 12-Lipoxygenase; 5-Lipoxygenase; 5-Lipoxygenase-activating protein; 5-Lipoxygenase-Activating Proteins - genetics; 5-Lipoxygenase-Activating Proteins - metabolism; antagonists; Arachidonate 5-Lipoxygenase - genetics; Arachidonate 5-Lipoxygenase - metabolism; Arachidonic acid; biosynthesis; Calcimycin - pharmacology; Calcium Ionophores - pharmacology; Cell Nucleus - enzymology; Cell Nucleus - genetics; cis-trans isomers; fluorescent antibody technique; HEK293 Cells; high performance liquid chromatography; Humans; Indoles - pharmacology; Leukotrienes - biosynthesis; Leukotrienes - genetics; Lipoxygenase Inhibitors - pharmacology; MK886; molecular biology; nuclear membrane; pharmacology; AA; 5-LO; 5-H(p)ETE; 12-LO; cPLA2; LT; Arachidonic acid; 5-Lipoxygenase-activating protein; HEK293 cells; FLAP; 5-Lipoxygenase; MK886; LTB4; 12-Lipoxygenase; HEK cells
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2014.05.016

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. 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. 5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca2+-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. 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. 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. •We established a HEK cell-based model with stably expressed 5-lipoxygenase (5-LO) ± five-lipoxygenase activating protein (FLAP).•FLAP co-expression stimulates the leukotriene biosynthesis by promoting conversion of 5-H(p)ETE to LTA4.•MK886 reduced leukotriene biosynthesis only in cell expressing 5-LO and FLAP.•Immunofluorescence showed co-localization of 5-LO and FLAP at the nuclear envelope upon A23187 stimulation.•This cell model will allow to study the 5-LO/FLAP interaction by mutations and putative FLAP inhibitors.