Evidence that increased 12-lipoxygenase activity induces apoptosis in fibroblasts.
The 12-lipoxygenase (LO) enzyme has been implicated in playing a role in pancreatic beta cell inflammatory damage and atherosclerosis. 12-LO reacts with fatty acids to form hydroperoxides which may alter cellular growth. In this study we investigated the direct effect of mouse leukocyte type 12-LO cDNA overexpression on apoptosis in Chinese hamster ovary fibroblast cells that also stably overexpress the angiotensin II type 1a receptor. CHO-AT1a cells expressing background levels of 12-LO exhibited clear increases in growth in response to angiotensin II. In contrast, the new 12-LO transfected cells (CHO-AT1a/ML12-LO cells) displayed reduced basal and angiotensin Il-induced growth compared to CHO-AT1a cells. Furthermore, serum-deprivation resulted in a significantly greater number of non-viable cells in clones having the greatest magnitude of 12-LO overexpression. These results suggested that reduction of the proliferation rate of CHO-AT1a/ML12-LO cells was due to an increasing rate of cell death. To determine whether the increase in cell death was due to apoptosis, we evaluated nuclear DNA fragmentation, cell morphologic changes, and activation of caspase-3. Cells overexpressing 12-LO cDNA displayed all these changes characteristic of apoptosis. In addition the 12-LO product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), directly induced apoptosis in CHO-AT1a cells. These results demonstrate for the first time that 12-LO activation can lead to apoptosis in fibroblasts, suggesting a role of 12-LO in leading to inflammatory mediated cellular damage.
Gu
J
J
Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Virginia, School of Medicine, MR 4 Building, Charlottesville, VA 22908, USA.
Liu
Y
Y
Wen
Y
Y
Natarajan
R
R
Lanting
L
L
Nadler
J L
JL
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D053938:DNA Fragmentation; D018076:DNA, Complementary; D007700:Kinetics; D015289:Leukotrienes / Q000494:pharmacology; D051379:Mice; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D015289:Leukotrienes; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D011994:Recombinant Proteins; D000804:Angiotensin II; C021201:12-HPETE; D001092:Arachidonate 12-Lipoxygenase; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases