Naveen Kaushal

Selenoprotein-dependent Up-regulation of Hematopoietic Prostaglandin D2 Synthase in Macrophages Is Mediated through the Activation of Peroxisome Proliferator-activated Receptor (PPAR) γ

The plasticity of macrophages is evident from their dual role in inflammation and resolution of inflammation that are accompanied by changes in the transcriptome and metabolome. Along these lines, we have previously demonstrated that the micronutrient selenium increases macrophage production of arachidonic acid (AA)-derived anti-inflammatory 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and decreases the proinflammatory PGE2. Here, we hypothesized that selenium modulated the metabolism of AA by a differential regulation of various prostaglandin (PG) synthases favoring the production of PGD2 metabolites, Δ12-PGJ2 and 15d-PGJ2. A dose-dependent increase in the expression of hematopoietic-PGD2 synthase (H-PGDS) by selenium and a corresponding increase in Δ12-PGJ2 and 15d-PGJ2 in RAW264.7 macrophages and primary bone marrow-derived macrophages was observed. Studies with organic non-bioavailable forms of selenium and the genetic manipulation of cellular selenium incorporation machinery indicated that selenoproteins were necessary for H-PGDS expression and 15d-PGJ2 production. Treatment of selenium-deficient macrophages with rosiglitazone, a peroxisome proliferator-activated receptor γ ligand, up-regulated H-PGDS. Furthermore, electrophoretic mobility shift assays indicated the presence of an active peroxisome proliferator-activated receptor-response element in murine Hpgds promoter suggesting a positive feedback mechanism of H-PGDS expression. Alternatively, the expression of nuclear factor-κB-dependent thromboxane synthase and microsomal PGE2 synthase was down-regulated by selenium. Using a Friend virus infection model of murine leukemia, the onset of leukemia was observed only in selenium-deficient and indomethacin-treated selenium-supplemented mice but not in the selenium-supplemented group or those treated with 15d-PGJ2. These results suggest the importance of selenium in the shunting of AA metabolism toward the production of PGD2 metabolites, which may have clinical implications.

Crucial Role of Macrophage Selenoproteins in Experimental Colitis

Inflammation is a hallmark of inflammatory bowel disease (IBD) that involves macrophages. Given the inverse link between selenium (Se) status and IBD-induced inflammation, our objective was to demonstrate that selenoproteins in macrophages were essential to suppress proinflammatory mediators, in part, by the modulation of arachidonic acid metabolism. Acute colitis was induced using 4% dextran sodium sulfate in wild-type mice maintained on Se-deficient (<0.01 ppm Se), Se-adequate (0.08 ppm; sodium selenite), and two supraphysiological levels in the form of Se-supplemented (0.4 ppm; sodium selenite) and high Se (1.0 ppm; sodium selenite) diets. Selenocysteinyl transfer RNA knockout mice (Trspfl/flLysMCre) were used to examine the role of selenoproteins in macrophages on disease progression and severity using histopathological evaluation, expression of proinflammatory and anti-inflammatory genes, and modulation of PG metabolites in urine and plasma. Whereas Se-deficient and Se-adequate mice showed increased colitis and exhibited poor survival, Se supplementation at 0.4 and 1.0 ppm increased survival of mice and decreased colitis-associated inflammation with an upregulation of expression of proinflammatory and anti-inflammatory genes. Metabolomic profiling of urine suggested increased oxidation of PGE2 at supraphysiological levels of Se that also correlated well with Se-dependent upregulation of 15-hydroxy-PG dehydrogenase (15-PGDH) in macrophages. Pharmacological inhibition of 15-PGDH, lack of selenoprotein expression in macrophages, and depletion of infiltrating macrophages indicated that macrophage-specific selenoproteins and upregulation of 15-PGDH expression were key for Se-dependent anti-inflammatory and proresolving effects. Selenoproteins in macrophages protect mice from dextran sodium sulfate–colitis by enhancing 15-PGDH–dependent oxidation of PGE2 to alleviate inflammation, suggesting a therapeutic role for Se in IBD.

Inhibition of TLR4-Induced IκB Kinase Activity by the RON Receptor Tyrosine Kinase and Its Ligand, Macrophage-Stimulating Protein

The RON receptor tyrosine kinase regulates the balance between classical (M1) and alternative (M2) macrophage activation. In primary macrophages, the ligand for Ron, macrophage-stimulating protein (MSP), inhibits the expression of inducible NO synthase, a marker of classically activated macrophages, whereas promoting the expression of arginase I, a marker of alternative activation. Ron−/− mice express increased levels of IL-12, a product of classically activated macrophages, after endotoxin administration, resulting in increased serum IFN-γ levels and enhanced susceptibility to septic shock. In this study, we demonstrate that MSP inhibits LPS-induced IL-12p40 expression, and this inhibition is dependent on the docking site tyrosines in Ron. To further define this inhibition, we examined the effect of Ron on signaling pathways downstream of Ron. We found that MSP does not inhibit the MyD88-independent activation of IFN regulatory factor 3 and production of IFN-β in response to LPS, nor does it inhibit MyD88-dependent TGF-β–activated kinase phosphorylation or MAPK activation in primary macrophages. However, the induction of IκB kinase activity, IκB degradation, and DNA binding of NF-κB after LPS stimulation is delayed in the presence of MSP. In addition, Ron inhibits serine phosphorylation of p65 and NF-κB transcriptional activity induced by LPS stimulation of TLR4. Finally, MSP inhibits the NF-κB–dependent upregulation of the nuclear IκB family member, IκBζ, a positive regulator of secondary response genes including IL-12p40. LPS also induces expression of Ron and an N-terminally truncated form of Ron, Sf-Ron, in primary macrophages, suggesting that the upregulation of Ron by LPS could provide classical feedback regulation of TLR signaling.

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.

Epigenetic regulation of inflammatory gene expression in macrophages by selenium

Acetylation of histone and non-histone proteins by histone acetyltransferases plays a pivotal role in the expression of proinflammatory genes. Given the importance of dietary selenium in mitigating inflammation, we hypothesized that selenium supplementation may regulate inflammatory gene expression at the epigenetic level. The effect of selenium towards histone acetylation was examined in both in vitro and in vivo models of inflammation by chromatin immunoprecipitation assays and immunoblotting. Our results indicated that selenium supplementation, as selenite, decreased acetylation of histone H4 at K12 and K16 in COX-2 and TNFα promoters, and of the p65 subunit of the redox sensitive transcription factor NFκB in primary and immortalized macrophages. On the other hand, selenomethionine had a much weaker effect. Selenite treatment of HIV-1-infected human monocytes also significantly decreased the acetylation of H4 at K12 and K16 on the HIV-1 promoter, supporting the down-regulation of proviral expression by selenium. A similar decrease in histone acetylation was also seen in the colonic extracts of mice treated with dextran sodium sulfate that correlated well with the levels of selenium in the diet. Bone-marrow-derived macrophages from Trsp(fl/fl)Cre(LysM) mice that lack expression of selenoproteins in macrophages confirmed the important role of selenoproteins in the inhibition of histone H4 acetylation. Our studies suggest that the ability of selenoproteins to skew the metabolism of arachidonic acid contributes, in part, to their ability to inhibit histone acetylation. In summary, our studies suggest a new role for selenoproteins in the epigenetic modulation of proinflammatory genes.

The regulation of erythropoiesis by selenium in mice.

Redox modulation by antioxidants, such as selenium (Se), has emerged as an important regulator of erythropoiesis. Using Se-deficient (0.04 ppm), Se-adequate (0.1 ppm), and Se-supplemented (0.4 ppm) C57/BL6 mice, we show that Se deficiency caused anemia, when compared to the Se-supplemented and Se-adequate groups. Increased denaturation of hemoglobin, methemoglobin, protein carbonyls, lipid peroxidation, Heinz bodies, and osmotic fragility of erythrocytes were observed in Se-deficient mice. Increased oxidative stress upregulated forkhead transcription factor (FoxO3a) and hypoxia-inducible factor-(HIF)1α in the spleen and kidney of Se-deficient murine as well as in the proerythroblast G1E cells cultured in Se-deficient media. A significant increase in the expression of erythropoietin, a downstream target of HIF1α, and expansion of stress erythroid progenitors (burst forming units-erythroid) were seen in the Se-deficient mice. Despite the increase in erythroid progenitors, lowered reticulocytes suggest a defective erythroid differentiation pathway. While Se deficiency led to increased nuclear levels of active FoxO3a, Se-adequate conditions reversed this effect and increased nuclear export by its binding partner, 14-3-3βζ, that is under the redox control of selenoproteins. In summary, these results provide insight into the importance of adequate Se nutrition in regulating red cell homeostasis by mitigating oxidative stress-dependent modulation of FoxO3a and HIF1α to effect differentiation of erythroid progenitors.

Selenium suppresses leukemia through the action of endogenous eicosanoids

Eradicating cancer stem-like cells (CSC) may be essential to fully eradicate cancer. Metabolic changes in CSC could hold a key to their targeting. Here, we report that the dietary micronutrient selenium can trigger apoptosis of CSC derived from chronic or acute myelogenous leukemias when administered at supraphysiologic but nontoxic doses. In leukemia CSC, selenium treatment activated ATM-p53-dependent apoptosis accompanied by increased intracellular levels of reactive oxygen species. Importantly, the same treatment did not trigger apoptosis in hematopoietic stem cells. Serial transplantation studies with BCR-ABL-expressing CSC revealed that the selenium status in mice was a key determinant of CSC survival. Selenium action relied upon the endogenous production of the cyclooxygenase-derived prostaglandins Δ(12)-PGJ2 and 15d-PGJ2. Accordingly, nonsteroidal anti-inflammatory drugs and NADPH oxidase inhibitors abrogated the ability of selenium to trigger apoptosis in leukemia CSC. Our results reveal how selenium-dependent modulation of arachidonic acid metabolism can be directed to trigger apoptosis of primary human and murine CSC in leukemia.

Evaluation of the Stability, Bioavailability, and Hypersensitivity of the Omega-3 Derived Anti-Leukemic Prostaglandin: Δ12-Prostaglandin J3

Previous studies have demonstrated the ability of an eicosapentaenoic acid (EPA)-derived endogenous cyclopentenone prostaglandin (CyPG) metabolite, Δ12-PGJ3, to selectively target leukemic stem cells, but not the normal hematopoietic stems cells, in in vitro and in vivo models of chronic myelogenous leukemia (CML). Here we evaluated the stability, bioavailability, and hypersensitivity of Δ12-PGJ3. The stability of Δ12-PGJ3 was evaluated under simulated conditions using artificial gastric and intestinal juice. The bioavailability of Δ12-PGJ3 in systemic circulation was demonstrated upon intraperitoneal injection into mice by LC-MS/MS. Δ12-PGJ3 being a downstream metabolite of PGD3 was tested in vitro using primary mouse bone marrow-derived mast cells (BMMCs) and in vivo mouse models for airway hypersensitivity. ZK118182, a synthetic PG analog with potent PGD2 receptor (DP)-agonist activity and a drug candidate in current clinical trials, was used for toxicological comparison. Δ12-PGJ3 was relatively more stable in simulated gastric juice than in simulated intestinal juice that followed first-order kinetics of degradation. Intraperitoneal injection into mice revealed that Δ12-PGJ3 was bioavailable and well absorbed into systemic circulation with a Cmax of 263 µg/L at 12 h. Treatment of BMMCs with ZK118182 for 12 h resulted in increased production of histamine, while Δ12-PGJ3 did not induce degranulation in BMMCs nor increase histamine. In addition, in vivo testing for hypersensitivity in mice showed that ZK118182 induces higher airways hyperresponsiveness when compared Δ12-PGJ3 and/or PBS control. Based on the stability studies, our data indicates that intraperitoneal route of administration of Δ12-PGJ3 was favorable than oral administration to achieve effective pharmacological levels in the plasma against leukemia. Δ12-PGJ3 failed to increase histamine and IL-4 in BMMCs, which is in agreement with reduced airway hyperresponsiveness in mice. In summary, our studies suggest Δ12-PGJ3 to be a promising bioactive metabolite for further evaluation as a potential drug candidate for treating CML.

OXIDATIVE STRESS AND INFLAMMATION: "THE LESSER OF TWO EVILS" IN CARCINOGENESIS

Literature is replete with reports indicating the involvement of oxidative stress and inflammation in tumorigenesis. Reactive oxygen species (ROS) are by-products of various oxidized cellular metabolites that serve as the “business end” as well as the consequence of oxidative stress leading to DNA damage, mutagenesis, and destruction of diverse biological macromolecules. These events may eventually culminate in carcinogenesisthrough varied mechanisms such as inhibition of apoptosis, increased cellular proliferation, angiogenesis, and disruption of DNA repair mechanism. Inflammation per se or as a ROSdependent mediator is a connecting link between carcinogenesis and oxidative insult. Alternatively, chronic inflammation via the exacerbation of ROS and pro-inflammatory cytokines aids in carcinogenesis by providing an environment conducive for exponential growth of malignant cells. Understanding the mechanisms of carcinogenesis over the years indicates that oxidative stress and inflammation as two distinct but related processes form the basis for the establishment of cancer, and/or aggressiveness to foster tumor growth the main sources of cancer. Though, these critical mediators of carcinogenesis are interrelated to each other, it is still not clear which leads to the other and contributes more towards to pathophysiology of cancer and. In this review, we describe the role of these processes in the complex and multifaceted mechanism of carcinogenesis. CARCINOGENESIS: CONCEPT AND CAUSES

Resolution of Cox mediated inflammation by Se supplementation in mouse experimental model of colitis

UC a form of IBD is a chronic inflammatory disorder of large intestine, with unknown etiology. Reports suggest a critical role of COX-2 dependent prostaglandins (PGs) mediated inflammatory pathway in pathophysiology of UC. However, COX inhibition using NSAIDs exacerbate IBD and thus is not a viable solution. Currently, in DSS induced experimental colitis in mice, we have demonstrated that dietary Se supplementation (0.5ppm as sodium selenite) symptomatically resolves the signs of inflammation in a redox sensitive manner as compared to Se deficient (0.01ppm) conditions, as seen by modulation in oxidative stress markers, morphological changes, histopathological examinations, biochemical studies such as MPO activity, activity of intestinal markers enzymes as well as mRNA and expressions of various pro and anti-inflammatory factors such as, mPGES, hPGDS, TXAS, 15-PGDH, GPX-1 and GPX-2. These findings were validated and correlated with changes in the biophysical parameters such as membrane fluidity, electrical parameters (impedance), transport across the colonic tissue and FTIR. Current study not only concluded that Se at supranutritional concentrations by modulating the redox status relieves the signs of colitis by regulating COX dependent PG biosynthetic pathway, but also sheds light on the biophysical characterization of these inflammatory/resolution pathways involved in UC.