Lipoxin generation by human megakaryocyte-induced 12-lipoxygenase.
Eicosanoid biosynthesis was examined with a human megakaryocytic cell line (Dami). Megakaryocytes incubated with [1-14C]arachidonic acid and either ionophore A23187 or thrombin generated both thromboxane and 12-hydroxyheptadecatrienoic acid (HHTrE). Exposure to phorbol myristate acetate (PMA) for 1 through 9 days induced differentiation and revealed an increase in the conversion of [1-14C]arachidonate to cyclooxygenase- and lipoxygenase (LO)-derived products. The LO-derived product was identified as 12S-HETE by its physical characteristics including GC/MS and chiral column SP-HPLC. PMA-treated Dami cells did not generate 5-HETE, leukotrienes or lipoxins from exogenous arachidonic acid while they did convert leukotriene A4 (LTA4) to lipoxin A4, lipoxin B4 and their respective all-trans isomers. In addition, COS-M6 cells transfected with a human 12-lipoxygenase cDNA and incubated with either arachidonic acid or LTA4 generated 12-HETE and lipoxins, respectively. The lipoxin profile generated by transfected COS-M6 cells incubated with LTA4 was similar to that generated by the PMA-treated Dami cells. Results indicate that human megakaryocytes can transform arachidonate and LTA4 to bioactive eicosanoids and that the 12-lipoxygenase appears upon further differentiation of these cells. In addition, they indicate that the 12-LO of human megakaryocytes and the 12-LO expressed by transfected COS cells can generate both lipoxins A4 and B4. Together they suggest that the human 12-LO can serve as a model of LX-synthetase activity with LTA4.
Sheppard
K A
KA
Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.
Greenberg
S M
SM
Funk
C D
CD
Romano
M
M
Serhan
C N
CN
D001092:Arachidonate 12-Lipoxygenase / Q000502:physiology*; D000001:Calcimycin / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis*; D017572:Leukotriene A4; D015289:Leukotrienes / Q000378:metabolism*; D008533:Megakaryocytes / Q000187:drug effects / Q000201:enzymology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D006893:Hydroxyeicosatetraenoic Acids; D017572:Leukotriene A4; D015289:Leukotrienes; D000001:Calcimycin; D001092:Arachidonate 12-Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate