Disruption of 12/15-Lipoxygenase Expression in Peritoneal Macrophages

Abstract

By in situ hybridization we show that the L-12LO gene is expressed abundantly in a subset of peritoneal macrophages but not in elicited leukocytes, alveolar macrophages, or bone marrow-derived macrophages. L-12LO is highly related to human and rabbit 15-lipoxygenases, enzymes that have been implicated in the maturation process of red blood cells, and the oxidative modification of low density lipoproteins that is implicated in atherogenesis. Accordingly, these enzymes have been referred to as 12/15-lipoxygenases. We have inactivated the L-12LO gene in mice using homologous recombination in embryonic stem cells. Macrophage expression of L-12LO was abolished in homozygous deficient mice as was formation of 12-hydroxyeicosatetraenoic acid (12-HETE). In zymosan-stimulated cells, there was significant diversion of metabolism to the 5-lipoxygenase products leukotriene C 4 and 5-HETE and in A23187-treated cells to 5-HETE only. The enhanced formation of 5-lipoxygenase metabolites was not due to compensatory changes of 5-lipoxygenase or 5-lipoxygenase activating protein but rather an apparent substrate diversion. L-12LO-deficient mice have no obvious abnormalities in reticulocyte or mature red blood cells, which suggest that in mice this pathway is not functionally important for erythrocytic development. Indices for oxidation of low density lipoprotein (measured as either thiobarbituric acid-reactive substances or the oxidant stress marker isoprostane 8-epi-prostaglandin F 2␣ ) were identical in incubations with unstimulated wild-type and L-12LO-deficient macrophages, but the zymosan-induced increase observed with wild-type macrophages was abolished in L-12LO-deficient cells. Thus, 12/15-lipoxygenase-deficient mice will be useful for the study of interaction between lipoxygenase pathways and determination of the in vivo role of 12/15-lipoxygenase-catalyzed oxidation of LDL in atherogenesis.12-Lipoxygenase incorporates stereospecifically molecular oxygen into arachidonic acid to form 12-hydroperoxyeicosatetraenoic acid (12-HPETE).1 Two types of 12-lipoxygenase enzymes have been described that differ biochemically and in immunoreactivity (1, 2). The primary sequences are 58 -65% identical based on the cloned cDNA sequence data (3-5). Enzyme activity for the "platelet-type" 12-lipoxygenase (P-12LO) was first found in human platelets more than 20 years ago (6). P-12LO has been found to be the predominant isoform in epidermal skin specimens and A431 epidermoid cells (5, 7-9). The other 12-lipoxygenase, referred to as "leukocyte-type," was first characterized extensively from porcine leukocytes (10) and was found to have a rather broad distribution in porcine and canine tissues by immunochemical assays (11,12). Besides tissue distribution, the leukocyte-type 12-lipoxygenase (L-12LO) is distinguished from the platelet-type enzyme by its ability to form 15-HPETE in addition to 12-HPETE from arachidonic acid substrate. L-12LO is highly related to 15LO in that both are dual specificity lipoxygenases, and they are about 85% identica...

14S,21R-Dihydroxydocosahexaenoic Acid Remedies Impaired Healing and Mesenchymal Stem Cell Functions in Diabetic Wounds

“…In murine skin wounds, there are other lipoxygenases, including epidermis-type LOX3, 12R-LOX, and 8-LOX as well as 5-LOX from recruited leukocytes (26,30,31,50,(53)(54)(55)(56)(57)(58)(59). However, LOX3, 12R-LOX, 8-LOX, or 5-LOX is unlikely to catalyze DHA 14S-hydroxygenation in skin wounds based on the study conducted by other scientists (52) and ourselves.…”

“…To identify dys-regulation in the formation of DHA-derived LMs in diabetic mice, we have conducted LC-MS/MS-based lipidomic studies (25) of wounded skin from diabetic db/db and nondiabetic db/ϩ mice. Here, we describe the identification of a novel DHA-derived mediator 14S,21R-diHDHA, which is suppressed in wounds of diabetic db/db mice and 12/15(or L-12)-lipoxygenase gene knock-out mice (12/15-LOX is most related to human 15-LOX type-1) (26). We demonstrate that 14S,21R-diHDHA restored wound healing and angiogenesis in diabetic mice as well as the pro-healing functions of db/db MSCs and key cellular processes of angiogenesis.…”

15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet

“…Alternatively, 15-LOX could participate in epididymal sperm maturation and particularly in the morphogenesis of the sperm tail midpiece and mitochondrial sheath. We are currently investigating epididymal sperm maturation in a mutant mouse lacking the 15-LOX gene (ALOX mouse; Sun & Funk 1996). Although the ALOX males are not infertile, our preliminary data indicate reduced sperm quality and altered CD migration (K W Lovercamp and P Sutovsky, unpublished observations).…”

12/15-Lipoxygenase Translocation Enhances Site-specific Actin Polymerization in Macrophages Phagocytosing Apoptotic Cells

“…However, the physiological role of 15-LO remains unclear. In reticulocytes it appears to play an important role in degradation of organelles as the cell matures, although no abnormality in red blood cell development was noted in the 12/15-LO Ϫ/Ϫ mice (27). Its physiological role in macrophages has remained equally obscure.…”