Role of human heme oxygenase-1 in attenuating TNF-alpha-mediated inflammation injury in endothelial cells.
Heme oxygenase (HO) is the rate-limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF-alpha (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO-1 gene exhibited a several-fold increase in HO-1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE(2) and 6-keto PGF(1alpha) levels. We also assessed the effect of retrovirus-mediated HO-1 gene transfer in the sense and antisense orientation on HO-1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO-1 sense and were greatly suppressed in cells transduced with HO-1 antisense as compared to control sham-transduced cells (P < 0.05). The percentage of the G(1)-phase in cells transduced with HO-1 significantly increased (41.4% +/- 9.1) compared with control endothelial cells (34.8% +/- 4.9). We measured COX activity by determining the levels of PGI(2) and PGE(2). The levels of PGI(2) decreased in cells transduced with HO-1 sense and increased in cells transduced with HO-1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO-1 sense and a marked increase in the HO-1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF-alpha-induced decrease in the proportion of G(1)-phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO-1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO-1. These results demonstrate that overexpressing HO-1 mitigated the TNF-alpha-mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity.
Kushida
Taketoshi
T
Department of Pharmacology, New York Medical College, Valhalla, New York 10595, USA.
Li Volti
Giovanni
G
Quan
Shuo
S
Goodman
Alvin
A
Abraham
Nader G
NG
D015121:6-Ketoprostaglandin F1 alpha / Q000096:biosynthesis; D017209:Apoptosis; D015153:Blotting, Western; D002453:Cell Cycle; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015232:Dinoprostone / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D016193:G1 Phase; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism* / Q000502:physiology*; D051547:Heme Oxygenase-1; D006801:Humans; D007249:Inflammation; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins; D008665:Metalloporphyrins / Q000494:pharmacology; D008840:Microfilament Proteins / Q000378:metabolism; D008954:Models, Biological; D009124:Muscle Proteins; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D008665:Metalloporphyrins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D016376:Oligonucleotides, Antisense; C058992:Tagln protein, mouse; D014409:Tumor Necrosis Factor-alpha; C055421:tin mesoporphyrin; D015121:6-Ketoprostaglandin F1 alpha; D004247:DNA; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone