Oxicams bind in a novel mode to the cyclooxygenase active site via a two-water-mediated H-bonding Network.

Oxicams are widely used nonsteroidal anti-inflammatory drugs (NSAIDs), but little is known about the molecular basis of the interaction with their target enzymes, the cyclooxygenases (COX). Isoxicam is a nonselective inhibitor of COX-1 and COX-2 whereas meloxicam displays some selectivity for COX-2. Here we report crystal complexes of COX-2 with isoxicam and meloxicam at 2.0 and 2.45 angstroms, respectively, and a crystal complex of COX-1 with meloxicam at 2.4 angstroms. These structures reveal that the oxicams bind to the active site of COX-2 using a binding pose not seen with other NSAIDs through two highly coordinated water molecules. The 4-hydroxyl group on the thiazine ring partners with Ser-530 via hydrogen bonding, and the heteroatom of the carboxamide ring of the oxicam scaffold interacts with Tyr-385 and Ser-530 through a highly coordinated water molecule. The nitrogen atom of the thiazine and the oxygen atom of the carboxamide bind to Arg-120 and Tyr-355 via another highly ordered water molecule. The rotation of Leu-531 in the structure opens a novel binding pocket, which is not utilized for the binding of other NSAIDs. In addition, a detailed study of meloxicam·COX-2 interactions revealed that mutation of Val-434 to Ile significantly reduces inhibition by meloxicam due to subtle changes around Phe-518, giving rise to the preferential inhibition of COX-2 over COX-1.

Effect of thrombin on human amnion mesenchymal cells, mouse fetal membranes, and preterm birth.

Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE2 in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE2 content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.

Vitamin D inhibits COX-2 expression and inflammatory response by targeting thioesterase superfamily member 4.

Inadequate vitamin D status has been linked to increased risk of type 2 diabetes and cardiovascular disease. Inducible cyclooxygenase (COX) isoform COX-2 has been involved in the pathogenesis of such chronic inflammatory diseases. We found that the active form of vitamin D, 1,25(OH)2D produces dose-dependent inhibition of COX-2 expression in murine macrophages under both basal and LPS-stimulated conditions and suppresses proinflammatory mediators induced by LPS. Administration of 1,25(OH)2D significantly alleviated local inflammation in a carrageenan-induced paw edema mouse model. Strikingly, the phosphorylation of both Akt and its downstream target IκBα in macrophages were markedly suppressed by 1,25(OH)2D in the presence and absence of LPS stimulation through up-regulation of THEM4 (thioesterase superfamily member 4), an Akt modulator protein. Knockdown of both vitamin D receptor and THEM4 attenuated the inhibitory effect of 1,25(OH)2D on COX-2 expression in macrophages. A functional vitamin D-responsive element in the THEM4 promoter was identified by chromatin immunoprecipitation and luciferase reporter assay. Our results indicate that vitamin D restrains macrophage-mediated inflammatory processes by suppressing the Akt/NF-κB/COX-2 pathway, suggesting that vitamin D supplementation might be utilized for adjunctive therapy for inflammatory disease.

Low molecular weight hyaluronan activates cytosolic phospholipase A2α and eicosanoid production in monocytes and macrophages.

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA2α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E2 production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA2α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE2 production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4(-/-) and Myd88(-/-) mice, but not in Cd44(-/-) mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA2α/COX2(high) and COX1/ALOX15/ALOX5/LTA4H(low) gene and PGE2/PGD2/15-HETE(high) and LXA4(low) eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.

Pre-existent asymmetry in the human cyclooxygenase-2 sequence homodimer.

Prostaglandin endoperoxide H synthase-2 (PGHS-2), also known as cyclooxygenase-2 (COX-2), is a sequence homodimer. However, the enzyme exhibits half-site heme and inhibitor binding and functions as a conformational heterodimer having a catalytic subunit (Ecat) with heme bound and an allosteric subunit (Eallo) lacking heme. Some recombinant heterodimers composed of a COX-deficient mutant subunit and a native subunit (i.e. Mutant/Native PGHS-2) have COX activities similar to native PGHS-2. This suggests that the presence of heme plus substrate leads to the subunits becoming lodged in a semi-stable Eallo-mutant/Ecat-Native∼heme form during catalysis. We examined this concept using human PGHS-2 dimers composed of combinations of Y385F, R120Q, R120A, and S530A mutant or native subunits. With some heterodimers (e.g. Y385F/Native PGHS-2), heme binds with significantly higher affinity to the native subunit. This correlates with near native COX activity for the heterodimer. With other heterodimers (e.g. S530A/Native PGHS-2), heme binds with similar affinities to both subunits, and the COX activity approximates that expected for an enzyme in which each monomer contributes equally to the net COX activity. With or without heme, aspirin acetylates one-half of the subunits of the native PGHS-2 dimer, the Ecat subunits. Subunits having an S530A mutation are refractory to acetylation. Curiously, aspirin acetylates only one-quarter of the monomers of S530A/Native PGHS-2 with or without heme. This implies that there are comparable amounts of two noninterchangeable species of apoenzymes, Eallo-S530A/Ecat-Native and Eallo-Native/Ecat-S530A. These results suggest that native PGHS-2 assumes a reasonably stable, asymmetric Eallo/Ecat form during its folding and processing.

Methionine adenosyltransferase II-dependent histone H3K9 methylation at the COX-2 gene locus.

BACKGROUND: MATII biosynthesizes AdoMet, which supplies methyl group for methylation of molecules, including histone. RESULTS: MATII interacts with histone methyltransferase SETDB1 and inhibits COX-2 gene expression. CONCLUSION: AdoMet synthesis and histone methylation are coupled on chromatin by a physical interaction of MATII and SETDB1 at the MafK target genes. SIGNIFICANCE: MATII may be important for both gene-specific and epigenome-wide regulation of histone methylation. Methionine adenosyltransferase (MAT) synthesizes S-adenosylmethionine (AdoMet), which is utilized as a methyl donor in transmethylation reactions involving histones. MATIIα, a MAT isozyme, serves as a transcriptional corepressor in the oxidative stress response and forms the AdoMet-integrating transcription regulation module, affecting histone methyltransferase activities. However, the identities of genes regulated by MATIIα or its associated methyltransferases are unclear. We show that MATIIα represses the expression of cyclooxygenase 2 (COX-2), encoded by Ptgs2, by specifically interacting with histone H3K9 methyltransferase SETDB1, thereby promoting the trimethylation of H3K9 at the COX-2 locus. We discuss both gene-specific and epigenome-wide functions of MATIIα.

15-hydroxyprostaglandin dehydrogenase-derived 15-keto-prostaglandin E2 inhibits cholangiocarcinoma cell growth through interaction with peroxisome proliferator-activated receptor-γ, SMAD2/3, and TAP63 proteins.

Prostaglandin E2 (PGE2) is a potent lipid mediator that plays a key role in inflammation and carcinogenesis. NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S)-hydroxyl group of PGE2, which leads to PGE2 biotransformation. In this study, we showed that the 15-PGDH-derived 15-keto-PGE2 is an endogenous peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand that causes PPAR-γ dissociation from Smad2/3, allowing Smad2/3 association with the TGF-ß receptor I and Smad anchor for receptor activation and subsequent Smad2/3 phosphorylation and transcription activation in human cholangiocarcinoma cells. The 15-PGDH/15-keto-PGE2-induced Smad2/3 phosphorylation resulted in the formation of the pSmad2/3-TAP63-p53 ternary complex and their binding to the TAP63 promoter, inducing TAP63 autotranscription. The role of TAP63 in 15-PGDH/15-keto-PGE2-induced inhibition of tumor growth was further supported by the observation that knockdown of TAP63 prevented 15-PGDH-induced inhibition of tumor cell proliferation, colony formation, and migration. These findings disclose a novel 15-PGDH-mediated 15-keto-PGE2 signaling cascade that interacts with PPAR-γ, Smad2/3, and TAP63.

Coordinate functional regulation between microsomal prostaglandin E synthase-1 (mPGES-1) and peroxisome proliferator-activated receptor γ (PPARγ) in the conversion of white-to-brown adipocytes.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE2. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE2 levels. In turn, PGE2 suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE2 on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B4). Taken together, these findings identify PGE2 as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.

Caveolin-1 interacts with Derlin-1 and promotes ubiquitination and degradation of cyclooxygenase-2 via collaboration with p97 complex.

Caveolin-1 (Cav-1) interacts with and mediates protein trafficking and various cellular functions. Derlin-1 is a candidate for the retrotranslocation channel of endoplasmic reticulum proteins. However, little is known about how Derlin-1 mediates glycosylated protein degradation. Here, we identified Cav-1 as a key player in Derlin-1- and p97-mediated cyclooxygenase 2 (COX-2) ubiquitination and degradation. Derlin-1 augmented the interaction of Cav-1 and COX-2 and mediated the degradation of COX-2 in a COX-2 C terminus-dependent manner. Suppression of Cav-1 decreased the ubiquitination of COX-2, and mutation of Asn-594 to Ala to disrupt N-glycosylation at the C terminus of COX-2 reduced the interaction of COX-2 with Cav-1 but not Derlin-1. Moreover, suppression of p97 increased the ubiquitination of COX-2 and up-regulated COX-2 but not COX-1. Cav-1 enhanced the interaction of p97 with Ufd1 and Derlin-1 and collaborated with p97 to interact with COX-2. Cav-1 may be a cofactor in the interaction of Derlin-1 and N-glycosylated COX-2 and may facilitate Derlin-1- and p97 complex-mediated COX-2 ubiquitination, retrotranslocation, and degradation.