C2-ceramide inhibits LPS-induced nitric oxide production in RAW 264.7 macrophage cells through down-regulating the activation of Akt.
ABSTRACT The effect of C(2)-ceramide, a membrane-permeable ceramide analogue, on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The non-toxic concentration of C(2)-ceramide inhibited LPS-induced NO production. It was due to the attenuated expression of the inducible type of NO synthase (iNOS). C(2)-ceramide did not influence the phosphorylation of a series of mitogen-activated protein (MAP) kinases in response to LPS. On the other hand, C(2)-ceramide down-regulated the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells, followed by the impairment of nuclear factor (NF)-kappaB activation. Moreover, the Akt dominant-negative mutant inhibited LPS-induced NO production. C(2)-ceramide was suggested to inhibit LPS-induced NO production through down-regulating the activation of Akt.
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ABSTRACT: The critical roles of kinase AKT in tumor cell proliferation, apoptosis and protein synthesis have been widely recognized. But AKT also plays an important role in the immune modulation. Recent studies have confirmed that kinase AKT can regulate the innate immune cell (neutrophil, macrophage and dendritic cell) development and functions. Studies have showed that different isoforms of kinase AKT have different effects in regulating immunity related diseases mainly through the mTOR dependent or independent pathway. The purpose of this review is just that illustrate the immune modulaton of kinase AKT on the innate immune cell development, survival and function. This article is protected by copyright. All rights reserved.Immunology 05/2013; DOI:10.1111/imm.12123 · 3.74 Impact Factor
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ABSTRACT: A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a twofold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.Analytical Chemistry 09/2013; 85(21). DOI:10.1021/ac401665u · 5.83 Impact Factor
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ABSTRACT: Abstract Ceramides play an essential role in modulating immune signaling pathways and pro-inflammatory cytokine production in response to infectious pathogens, stress stimuli, or chemotherapeutic drugs. In this study, we demonstrated that Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), the pathogen for aggressive periodontitis, induced de novo synthesis of ceramide in Raw 264.7 cells. In addition, we identified that fenretinide, a synthetic retinoid, suppressed the de novo synthesis of ceramide induced by A. actinomycetemcomitans. Moreover, fenretinide attenuated interleukin (IL)-1β, IL-6 and cyclooxygenase (COX)-2 mRNA expression induced by A. actinomycetemcomitans. Fenretinide also decreased IL-1β, IL-6, and prostaglandin E2 pro-inflammatory cytokine levels in Raw 264.7 cells induced by A. actinomycetemcomitans. However, fenretinide had no significant effects on TNF-α mRNA or protein levels. Furthermore, we showed that fenretinide inhibited the JAK-STAT, PI3K-Akt, PKC and NF-κB signaling pathways, whereas fenretinide up-regulated MAPK signaling pathways after bacterial stimulation. In conclusion, this study emphasizes the de novo ceramide synthesis pathway in response to bacterial stimulation and demonstrates the anti-inflammatory role of fenretinide in bacteria-induced immune response.The Journal of Lipid Research 11/2012; DOI:10.1194/jlr.M031427 · 4.73 Impact Factor