The anti-inflammatory effects of selenium are mediated through 15-deoxy-Delta12,14-prostaglandin J2 in macrophages.

Selenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-kappaB-dependent pro-inflammatory gene expression. To understand the molecular mechanisms underlying the anti-inflammatory property of selenium, we examined the activity of a key kinase of the NF-kappaB cascade, IkappaB-kinase beta (IKKbeta) subunit, as a function of cellular selenium status in murine primary bone marrow-derived macrophages and RAW264.7 macrophage-like cell line. In vitro kinase assays revealed that selenium supplementation decreased the activity of IKKbeta in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of selenium-supplemented macrophages resulted in a time-dependent increase in 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) formation, an endogenous inhibitor of IKKbeta activity. Further analysis revealed that inhibition of IKKbeta activity in selenium-supplemented cells correlated with the Michael addition product of 15d-PGJ2 with Cys-179 of IKKbeta, while the formation of such an adduct was significantly decreased in the selenium-deficient macrophages. In addition, anti-inflammatory activities of selenium were also mediated by the 15d-PGJ2-dependent activation of the peroxisome proliferator-activated nuclear receptor-gamma in macrophages. Experiments using specific cyclooxygenase (COX) inhibitors and genetic knockdown approaches indicated that COX-1, and not the COX-2 pathway, was responsible for the increased synthesis of 15d-PGJ2 in selenium-supplemented macrophages. Taken together, our results suggest that selenium supplementation increases the production of 15d-PGJ2 as an adaptive response to protect cells against oxidative stress-induced pro-inflammatory gene expression. More specifically, modification of protein thiols by 15d-PGJ2 represents a previously undescribed code for redox regulation of gene expression by selenium.

Selenium deficiency increases the expression of inducible nitric oxide synthase in RAW 264.7 macrophages: role of nuclear factor-kappaB in up-regulation.

The inducible isoform of nitric oxide synthase (iNOS) is implicated in atherosclerosis, malignancy, rheumatoid arthritis, tissue and reperfusion injuries. A key determinant of the pro-oxidant versus protective effects of NO is the underlying redox status of the tissue. Selenoproteins, such as glutathione peroxidases (GPxs) and thioredoxin reductases, are key components of cellular defence and promote optimal antioxidant/oxidant balance. In this study, we have investigated the relationship between Se status, iNOS expression and NO production in Se-deficient and Se-supplemented RAW 264.7 macrophage cell lines. The cellular GPx activity, a measure of Se status, was 17-fold lower in Se-deficient RAW 264.7 cells and the total cellular oxidative tone, as assessed by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate, was higher in the Se-deficient cells than the Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation of these cells in culture, we found significantly higher iNOS transcript and protein expression levels with an increase in NO production in Se-deficient RAW 264.7 cells than the Se-supplemented cells. Electrophoretic mobility-shift assays, nuclear factor-kappaB (NF-kappaB)-luciferase reporter assays and Western blot analyses indicate that the increased expression of iNOS in Se deficiency could be due to an increased activation and consequent nuclear localization of the redox-sensitive transcription factor NF-kappaB. These results suggest an inverse relationship between cellular Se status and iNOS expression in LPS-stimulated RAW 264.7 cells and provide evidence for the beneficial effects of dietary Se supplementation in the prevention and/or treatment of oxidative-stress-mediated inflammatory diseases.

Inhibition of potato lipoxygenase by linoleyl hydroxamic acid: kinetic and EPR spectral evidence for a two-step reaction.

The reaction mechanism of an electrophoretically pure potato tuber lipoxygenase (ptLOX) was studied by EPR spectroscopy. An EPR spectrum of the 'native' ptLOX recorded at 4.5+/-0.5 K showed signals of a high-spin (pseudo) axial Fe(3+) with a g-value of approx. 6.3+/-0.1 with a shoulder at g=5.9+/-0.1, and a rhombic Fe(3+) signal at g=4.35+/-0.05. When the enzyme was treated with a 2-fold molar excess of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE], a 3-fold increase in the integral intensity of the g=6.3 signal was observed, indicating that 25% of the native ptLOX iron was in ferrous state. The positional isomer 9(S)-HPODE caused similar spectral changes. Therefore the catalytic centre of ptLOX appears to accommodate both positional isomers of linoleic acid hydroperoxides in a manner that ensures proper alignment of their hydroperoxy groups with the iron centre of the enzyme. Treatment of the Fe(3+)-ptLOX form with a 3-fold molar excess of linoleyl hydroxamic acid (LHA) completely quenched the g=6.3 signal. Concurrently, a dramatic increase in the signal at g=4.35 was detected, which was attributed to a newly formed LHA-Fe(3+)-ptLOX complex. The spectral characteristics of the complex are similar to those of a 4-nitrocatechol-Fe(3+)-ptLOX complex. From these observations, we conclude that LHA did not reduce Fe(3+) to Fe(2+), but rather formed a LHA-Fe(3+)-ptLOX complex. Formation of such a complex may be responsible for the inhibitory activity of LHA, at least in the initial stages of enzyme inhibition. A prolonged 15 min incubation of the complex at 23+/-1 degrees C led to the partial quenching of the g=4.35 signal. The quenching is attributed to the reduction of Fe(3+)-ptLOX by LHA, with concomitant formation of its oxidation product(s). A kinetic scheme for the inhibition is proposed.

Nuclear factor-kappaB mediates over-expression of cyclooxygenase-2 during activation of RAW 264.7 macrophages in selenium deficiency.

Selenium (Se) is an essential micronutrient for all mammalian species and is associated with a variety of physiological functions, notably immune system, in the form of selenoproteins. Inadequate Se nutrition has been linked to various diseases, including rheumatoid arthritis, cardiomyopathy, and cancer. Important to this discussion is that cyclooxygenase-2 (COX-2) is over-expressed in all the aforesaid pathologies; however, a casual relationship between Se status and COX-2 expression remains to be established. The present study is based on the hypothesis that oxidant stress, a consequence of Se deficiency, lowers the activation potential of the redox-sensitive transcription factor, NF-kappaB, and that the activated NF-kappaB is required for the altered expression of COX-2. To test this hypothesis, we have investigated the relationship between Se status and COX-2 expression in response to LPS stimulation in RAW 264.7, a macrophage-like cell line. In Se-deficient cells, the Se-dependent glutathione peroxidase activity (Se-GPx), a measure of Se status, was markedly reduced and the overall oxidative stress was significantly higher than Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation, we found 2-3-folds higher COX-2 protein expression as well as higher PGE2 levels in Se-deficient cells than Se-supplemented cells. In comparison, COX-1 protein expression was not affected by either LPS stimulation or Se status. Following LPS stimulation, the nuclear localization of NF-kappaB was significantly increased in Se-deficient macrophages, thereby leading to increased expression of COX-2. This is the first report demonstrating an inverse relationship between Se status and the expression of COX-2.