Melatonin suppresses macrophage cyclooxygenase-2 and inducible nitric oxide synthase expression by inhibiting p52 acetylation and binding

Abstract

Melatonin has been shown to be produced by nonpineal cells and possess anti-inflammatory actions in animal models. In the present study, we tested the hypothesis that melatonin suppresses the expression of proinflammatory genes such as cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (INOS) by a common transcriptional mechanism. Melatonin but not tryptophan or serotonin inhibited lipopolysaccharide (LPS)-induced COX-2 and iNOS protein levels and promoter activities in RAW 264.7 cells in a time-and concentration-dependent manner. LPS or LPS plus interferon-␥ (IFN␥) increased binding of all 5 isoforms of NF-B to COX-2 and iNOS promoters. Melatonin selectively inhibited p52 binding without affecting p100 expression, p52 generation from p100, or p52 nuclear translocation. p52 acetylation was enhanced by LPS, which was abrogated by melatonin. Melatonin inhibited p300 histone acetyltransferase (HAT) activity and abrogated p300-augmented COX-2 and iNOS expression. HAT inhibitors suppressed LPS-induced p52 binding and acetylation to an extent similar to melatonin, and melatonin did not potentiate the effect of HAT inhibitors. These results suggest that melatonin inhibits COX-2 and iNOS transcriptional activation by inhibiting p300 HAT activity, thereby suppressing p52 acetylation, binding, and transactivation. ( IntroductionMelatonin is produced primarily in the pineal gland and secreted into circulating blood. It has been shown that melatonin is also produced by cells including monocytes and macrophages and has been implicated in protection against inflammation (for a review, see Reiter 1 ). Depletion of endogenous melatonin has been shown in rats to aggravate carrageenan-induced pleurisy, which is reversed by exogenous melatonin, 2 while exogenous melatonin administration has been demonstrated to reduce acute inflammatory reactions in zymosan-activated plasma-induced paw inflammation 3 and carrageenan-induced edema and pleurisy. 4 Its anti-inflammatory actions are attributed to its ability to scavenge reactive oxygen species and suppress proinflammatory genes, including inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX2). 5,6 iNOS (also known as NOS-2) and COX-2 play an important role in diverse inflammatory conditions including vascular inflammation, atherosclerosis, and plaque stability. 7-10 iNOS catalyzes the robust synthesis of nitric oxide (NO) from L-arginine. 11 NO induces inflammatory reactions and tissue injury by diverse mechanisms including activation of soluble guanylyl cyclase, protein nitration, and nitrosylation. 12-14 COX-2 occupies a key position in production of proinflammatory prostanoids such as prostaglandin E 2 (PGE 2 ). 15 iNOS and COX-2 share similar cellular and molecular properties. 16 Their expressions are highly inducible by cytokines and lipopolysaccharide (LPS). [17][18][19][20] They are coinduced in inflammatory cells and their products work in concert to cause tissue inflammation and damage. 7 Transcriptional regulation of COX2 and INOS in murine RAW 264.7 ce...

Tumor-specific Activation of Human Telomerase Reverses Transcriptase Promoter Activity by Activating Enhancer-binding Protein-2β in Human Lung Cancer Cells

“…1) The binding of other AP-2 isoforms such as AP-2␣ was not different between lung cancer cell lines and normal lung cells; 2) the binding of the protein complex was undetectable when the AP-2 sites in the hTERT promoter were mutated or a non-relevant probe was used; and 3) neither nonimmune IgG nor anti-von Willebrand factor antibody detected any protein that was pulled down by the streptavidin-agarose beads. Although EMSA is conventionally used to qualitatively assess DNA-protein complex formation, this technique is inadequate for identifying the individual proteins in a large complex because the DNA-protein complex is too large to be resolved by gel electrophoresis (40). The streptavidin-agarose pulldown assay has recently been used successfully to analyze the specific binding of transactivators and coactivators to the cancer-related genes COX-2 and NOS-2 (39,40,43).…”

“…Although EMSA is conventionally used to qualitatively assess DNA-protein complex formation, this technique is inadequate for identifying the individual proteins in a large complex because the DNA-protein complex is too large to be resolved by gel electrophoresis (40). The streptavidin-agarose pulldown assay has recently been used successfully to analyze the specific binding of transactivators and coactivators to the cancer-related genes COX-2 and NOS-2 (39,40,43). The method allows the rapid, sensitive, and scalable detection of relative binding activities and specificities directly from cellular extracts.…”

“…Chromatin Immunoprecipitation (ChIP)-The ChIP assay was done as described previously (39,40). Briefly, 1% formaldehyde was added to the culture medium; after incubation for 20 min at 37°C, the cells were washed twice with PBS, scraped, and lysed for 10 min at 4°C in lysis buffer (10 mM Tris-HCl (pH 8.0), 1% SDS, 1 mM phenylmethylsulfonyl fluoride, pepstatin A, and aprotinin).…”

“…DNA-Protein Binding Assay-DNA-protein binding assays were performed using a streptavidin-agarose bead pulldown assay as described previously (39,40). Briefly, 80 -90% confluent cells were harvested, and nuclear extracts were prepared.…”

Melatonin: A pleiotropic molecule regulating inflammation

“…After experimental inflammation induction in vivo and ex vivo, melatonin was shown to prevent or reduce the inflammatory-derived activation of PLA2 [88] , LOX [89] and COX [90]. The reduction of PLA2 [88] and LOX [89] up-regulation by melatonin occurs via nonoxidative mechanisms, implying the engagement of MT1/MT2 [88] or ROR/RZR [89] receptors; thus, many different actions of melatonin including receptor stimulation or radical scavenging seem to cooperate to achieve the same final goal, that is to interfere with the inflammatory cascade.…”