Ryan J. Arner

The anti-inflammatory effects of selenium are mediated through 15-Deoxy-Δ12, 14-prostaglandin J2 in macrophages

Selenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-κB-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-κB cascade, IκB-kinase β (IKKβ) 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 IKKβ in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of selenium-supplemented macrophages resulted in a time-dependent increase in 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) formation, an endogenous inhibitor of IKKβ activity. Further analysis revealed that inhibition of IKKβ activity in selenium-supplemented cells correlated with the Michael addition product of 15d-PGJ2 with Cys-179 of IKKβ, 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-γ 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 attenuates pro-inflammatory gene expression in macrophages.

Selenium (Se) is an important element required for the optimal functioning of the immune system. Particularly in macrophages, which play a pivotal role in immune regulation, Se acts as a major antioxidant in the form of selenoproteins to mitigate the cytotoxic effects of reactive oxygen species. Here we describe the role of Se as an anti-inflammatory agent and its effect on the macrophage signal transduction pathways elicited by bacterial endotoxin, LPS. Our studies demonstrate that supplementation of Se to macrophages (Se-deficient) leads to a significant decrease in the LPS-induced expression of two important pro-inflammatory genes, cyclooxygenase-2 (COX-2) and tumor necrosis factor-alpha (TNF-alpha) via the inhibition of MAP kinase pathways. Furthermore, Se-deficiency in mice exacerbated the LPS-mediated infiltration of macrophages into the lungs suggesting that Se status is a crucial host factor that regulates inflammation. In summary, our results indicate that Se plays an important role as an anti-inflammatory agent by tightly regulating the expression of pro-inflammatory genes in immune cells.

myo-Inositol oxygenase: molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and d-chiro-inositol

myo-Inositol oxygenase (MIOX) catalyses the first committed step in the only pathway of myo-inositol catabolism, which occurs predominantly in the kidney. The enzyme is a non-haem-iron enzyme that catalyses the ring cleavage of myo-inositol with the incorporation of a single atom of oxygen. A full-length cDNA was isolated from a pig kidney library with an open reading frame of 849 bp and a corresponding protein subunit molecular mass of 32.7 kDa. The cDNA was expressed in a bacterial pET expression system and an active recombinant MIOX was purified from bacterial lysates to electrophoretic homogeneity. The purified enzyme displayed the same catalytic properties as the native enzyme with K(m) and k(cat) values of 5.9 mM and 11 min(-1) respectively. The pI was estimated to be 4.5. Preincubation with 1 mM Fe(2+) and 2 mM cysteine was essential for the enzymes activity. D-chiro-Inositol, a myo-inositol isomer, is a substrate for the recombinant MIOX with an estimated K(m) of 33.5 mM. Both myo-inositol and D-chiro-inositol have been implicated in the pathogenesis of diabetes. Thus an understanding of the regulation of MIOX expression clearly represents a potential window on the aetiology of diabetes as well as on the control of various intracellular phosphoinositides and key signalling pathways.

Gambogic acid covalently modifies IκB kinase-β subunit to mediate suppression of lipopolysaccharide-induced activation of NF-κB in macrophages

GA (gambogic acid) is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi with a long history of use as a complementary and alternative medicine. The antitumour activity of GA has been well demonstrated and is thought to arise partly from the associated anti-inflammatory activity. Recent studies have indicated that the antitumour activity of GA is mediated by its ligation of TfR1 (transferrin receptor-1). Since the cellular expression of TfR1 is down-regulated by LPS (lipopolysaccharide), we hypothesized that an alternative pathway exists in immune cells, such as macrophages, where GA could mitigate the expression of pro-inflammatory genes. Here we demonstrate that GA inhibits the LPS-dependent expression of NF-kappaB (nuclear factor kappaB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-kappaB-luciferase reporter and gel-shift analyses revealed that GA strongly blocked the activation of NF-kappaB induced by LPS, whereas 9,10-dihydro-GA, which lacks the reactive alpha,beta-unsaturated carbonyl group, was ineffective. Moreover, GA was able to decrease nuclear p65 levels in RAW264.7 macrophages, where the expression of TfR1 was down-regulated by RNA interference. in vitro kinase assays coupled with interaction studies using biotinylated GA as well as proteomic analysis demonstrated that IKKbeta [IkappaB (inhibitory kappaB) kinase-beta], a key kinase of the NF-kappaB signalling axis, was covalently modified by GA at Cys-179, causing significant inhibition of its kinase activity. Taken together, these results demonstrate the potent anti-inflammatory activity of GA.

Synthesis and evaluation of the anti-inflammatory properties of selenium-derivatives of celecoxib

Celecoxib is a selective cyclooxygenase (COX)-2 inhibitor used to treat inflammation, while selenium is known to down-regulate the transcription of COX-2 and other pro-inflammatory genes. To expand the anti-inflammatory property, wherein celecoxib could inhibit pro-inflammatory gene expression at extremely low doses, we incorporated selenium (Se) into two Se-derivatives of celecoxib, namely; selenocoxib-2 and selenocoxib-3. In vitro kinetic assays of the inhibition of purified human COX-2 activity by these compounds indicated that celecoxib and selenocoxib-3 had identical K(I) values of 2.3 and 2.4μM; while selenocoxib-2 had a lower K(I) of 0.72μM. Furthermore, selenocoxib-2 inhibited lipopolysaccharide-induced activation of NF-κB leading to the down-regulation of expression of COX-2, iNOS, and TNFα more effectively than selenocoxib-3 and celecoxib in RAW264.7 macrophages and murine bone marrow-derived macrophages. Studies with rat liver microsomes followed by UPLC-MS-MS analysis indicated the formation of selenenylsulfide conjugates of selenocoxib-2 with N-acetylcysteine. Selenocoxib-2 was found to release minor amounts of Se that was effectively inhibited by the CYP inhibitor, sulphaphenazole. While these studies suggest that selenocoxib-2, but not celecoxib and selenocoxib-3, targets upstream events in the NF-κB signaling axis, the ability to effectively suppress NF-κB activation independent of cellular selenoprotein synthesis opens possibilities for a new generation of COX-2 inhibitors with significant and broader anti-inflammatory potential.