The enzymatic machinery of leukotriene biosynthesis Studies on ontogenic expression, interactions and function

Sammanfattning: Leukotrienes (LTs) are biologically active arachidonic acid (AA) derivatives generated by the 5-lipoxygenase (5-LO) pathway. They are produced by myeloid cells. 5-LO converts AA to LTA4 in cooperation with 5-LO activating protein (FLAP). LTA4 is converted to LTB4, by LTA4-hydrolase (LTA4H) or to LTC4 by LTC4-synthase (LTC4S). LTs act on cells through plasma membrane bound G-protein coupled receptors found on leukocytes, smooth muscle and endothelial cells. We report here protein-protein interactions of proteins involved in LTC4 synthesis. 5-LO interacts with cytosolic domains of the integral membrane proteins FLAP and LTC4S at the nuclear envelope, in addition LTC4S interacts with FLAP through its hydrophobic membrane spanning regions. We constructed an LTC4S promoter controlled GFP reporter vector, displaying cell specific expression and sensitivity to agents known to affect LTC4S expression. The vector was used to create transgenic mice expressing GFP as a reporter for LTC4S. Ontogenic mouse expression studies revealed that the complete LT biosynthesis machinery was present at e11.5 primarily in the hematopoietic cells colonizing the liver. Although mature myeloid cells were the main contributors, a substantial amount of FLAP message was also detected in hematopoietic stem and progenitor cells, indicating possible functions for FLAP in hematopoietic regulation. Functional analyses using FLAP knockout mice suggested fine-tuning roles for LTs during differentiation, primarily along the B-lymphocyte differentiation path.