Protein kinase B in the diabetic heart

Cytosolic phospholipases A₂ (cPLA₂) and cyclooxygenases-1 and -2 (COX-1 and -2) play a pivotal role in the metabolism of arachidonic acid (AA) and in eicosanoid production. The coordinate regulation and expression of these enzymes is not well defined. In this study, the effect of phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor (TNF), lipopolysaccharide (LPS) and macrophage-colony stimulating factor (M-CSF) on AA release and prostaglandin E₂ (PGE₂) production and the expression of cPLA₂ and COX-1 and -2 were investigated in U937 human pre-monocytic cells and fully differentiated macrophages. Treatment of U937 cells with PMA or macrophages with LPS increased AA release and PGE₂ production. Incubation of U937 cells or macrophages for 8 h with all stimuli elevated cPLA₂ expression. In contrast, cPLA₂ expression was reduced upon further incubation of U937 cells or macrophages for 24 h with all stimuli indicating a bi-phasic expression pattern of this enzyme. PMA induced COX-1 expression in U937 cells whereas LPS induced COX-2 expression in macrophages. Although TNF and M-CSF induced a significant amount of AA release in both cell models, they failed to induce a comparable production of PGE₂ since they were unable to induce the coordinate expression of the downstream key enzymes, COX-1 or COX-2. The results suggest that the enhancement of AA release in both U937 cells and macrophages may be caused by both increased cPLA₂ activity and elevated cPLA₂ protein expression. In addition, PMA stimulates PGE₂ production via up-regulation of COX-1, and likely COX-2, expression in U937 cells whereas LPS stimulates PGE₂ production via induction of COX-2 expression in macrophages.     

An accessory role for ceramide in interleukin-1β induced prostaglandin synthesis

Interleukin-1 (IL-1) is a potent inducer of prostaglandin E₂ (PGE₂) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE₂ production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE₂ production by examining their respective effects on the rate-limiting enzymes in PGE₂ synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A₂ (cPLA₂). IL-1-induced PGE₂ synthesis required 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL- 1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA₂ mRNA and protein expression which corresponded with the initiation of PGE₂ synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL- 1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE₂ synthesis required both COX-2 and cPLA₂ protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein accumulation resulting in enhanced PGE₂ production.     

Retinoic acid increases hypoxia-inducible factor-1α through intracrine prostaglandin E2 signaling in human renal proximal tubular cells HK-2

We have previously shown in HK-2 cells that ATRA (all-trans-retinoic acid) up-regulates HIF-1α (hypoxia-inducible factor-1α) in normoxia, which results in increased production of renal protector VEGF-A (vascular endothelial growth factor-A). Here we investigated the role of COXs (cyclooxygenases) in these effects and we found that, i) ATRA increased the expression of COX-1 and COX-2 mRNA and protein and the intracellular levels (but not the extracellular ones) of PGE₂. Furthermore, inhibitors of COX isoenzymes blocked ATRA-induced increase in intracellular PGE₂, HIF-1α up-regulation and increased VEGF-A production. Immunofluorescence analysis found intracellular staining for EP1-4 receptors (PGE₂ receptors). These results indicated that COX activity is critical for ATRA-induced HIF-1α up-regulation and suggested that intracellular PGE₂ could mediate the effects of ATRA; ii) Treatment with PGE₂ analog 16,16-dimethyl-PGE₂ resulted in up-regulation of HIF-1α and antagonists of EP1-4 receptors inhibited 16,16-dimethyl-PGE₂- and ATRA-induced HIF-1α up-regulation. These results confirmed that PGE₂ mediates the effects of ATRA on HIF-1α expression; iii) Prostaglandin uptake transporter inhibitor bromocresol green blocked the increase in HIF-1α expression induced by PGE₂ or by PGE₂-increasing cytokine interleukin-1β, but not by ATRA. Therefore only intracellular PGE₂ is able to increase HIF-1α expression. In conclusion, intracellular PGE₂ increases HIF-1α expression and mediates ATRA-induced HIF-1α up-regulation.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

Resveratrol inhibits urban particulate matter-induced COX-2/PGE2 release in human fibroblast-like synoviocytes via the inhibition of activation of NADPH oxidase/ROS/NF-κB

Human fibroblast-like synoviocytes (FLSs) play a role in joint synovial inflammation in rheumatoid arthritis (RA). Some evidence indicates that particulate matter (PM) in air pollution could contribute to the progression of RA. However, more research is needed to clarify this relationship. Up-regulation of cyclooxygenase (COX)-2 and its metabolite prostaglandin E₂ (PGE₂) are implicated in various inflammatory diseases. Resveratrol, a polyphenol found mainly in grapes and red wine, has antioxidant and anti-inflammatory activities. In the present study, we demonstrated that resveratrol reduced PM-induced COX-2/PGE₂ expression in human FLSs, and attenuated PM-enhanced NADPH oxidase activity and ROS generation. In addition, PM induced Akt, ERK1/2, or p38 MAPK activation, which was inhibited by resveratrol. Finally, we demonstrated that PM enhanced NF-κB p65 phosphorylation and the NF-κB promoter activity, which were reduced by pretreatment with a ROS inhibitor or resveratrol. Thus, we concluded that resveratrol functions as a suppressor of PM-induced inflammatory signaling pathways by inhibiting COX-2/PGE₂ expression.     

Autocrine prostaglandin E2 signaling promotes promonocytic leukemia cell survival via COX-2 expression and MAPK pathway

The COX-2/PGE₂ pathway has been implicated in the occurrence and progression of cancer. The underlying mechanisms facilitating the production of COX-2 and its mediator, PGE₂, in cancer survival remain unknown. Herein, we investigated PGE₂-induced COX-2 expression and signaling in HL-60 cells following menadione treatment. Treatment with PGE₂ activated anti-apoptotic proteins such as Bcl-2 and Bcl-xL while reducing pro-apoptotic proteins, thereby enhancing cell survival. PGE₂ not only induced COX-2 expression, but also prevented casapse-3, PARP, and lamin B cleavage. Silencing and inhibition of COX-2 with siRNA transfection or treatment with indomethacin led to a pronounced reduction of the extracellular levels of PGE₂, and restored the menadione-induced cell death. In addition, pretreatment of cells with the MEK inhibitor PD98059 and the PKA inhibitor H89 abrogated the PGE₂-induced expression of COX-2, suggesting involvement of the MAPK and PKA pathways. These results demonstrate that PGE₂ signaling acts in an autocrine manner, and specific inhibition of PGE₂ will provide a novel approach for the treatment of leukemia. [BMB Reports 2015; 48(2): 109-114]     

Prostaglandin E synthases: Understanding their pathophysiological roles through mouse genetic models

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin H₂ (PGH₂) to PGE₂, is known to comprise a group of at least three structurally and biologically distinct enzymes. Two of them are membrane-bound and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli and downregulated by anti-inflammatory glucocorticoids as in the case of COX-2. It is functionally coupled with COX-2 in marked preference to COX-1. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE₂ production. Recently, mice have been engineered with specific deletions in each of these three PGES enzymes. In this review, we summarize the current understanding of the in vivo roles of PGES enzymes by knockout mouse studies and provide an overview of their biochemical properties.     

Prostaglandin E2/EP1 Signaling Pathway Enhances Intercellular Adhesion Molecule 1 (ICAM-1) Expression and Cell Motility in Oral Cancer Cells

Oral squamous cell carcinoma has a striking tendency to migrate and metastasize. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. However, the effects of COX-2 on human oral cancer cells are largely unknown. We found that overexpression of COX-2 or exogenous PGE₂ increased migration and intercellular adhesion molecule 1 (ICAM)-1 expression in human oral cancer cells. Using pharmacological inhibitors, activators, and genetic inhibition of EP receptors, we discovered that the EP1 receptor, but not other PGE receptors, is involved in PGE₂-mediated cell migration and ICAM-1 expression. PGE₂-mediated migration and ICAM-1 up-regulation were attenuated by inhibitors of protein kinase C (PKC)δ, and c-Src. Activation of the PKCδ, c-Src, and AP-1 signaling pathway occurred after PGE₂ treatment. PGE₂-induced expression of ICAM-1 and migration activity were inhibited by a specific inhibitor, siRNA, and mutants of PKCδ, c-Src, and AP-1. In addition, migration-prone sublines demonstrated that cells with increased migration ability had higher expression of COX-2 and ICAM-1. Taken together, these results indicate that the PGE₂ and EP1 interaction enhanced migration of oral cancer cells through an increase in ICAM-1 production.     

Up-regulation of microsomal prostaglandin E synthase-1 in COX-1 and COX-2 knock-out mouse fibroblast cell lines

In this paper we investigated the possible involvement of prostaglandin E synthases (PGESs) in compensatory mechanism. Our findings showed that microsomal (m)PGES-1 expression was significantly up-regulated in COX knock-out (K/O) cells whereas the expression of cytosolic PGES was not changed indicating that the induction of mPGES-1 may, at least in part, contribute to the substantial increase of PGE₂ production in COX K/O cell lines. The selective up-regulation of mPGES-1 in COX-2 K/O cells suggests that mPGES-1 may be metabolically coupled with COX-1 for PGE₂ formation. Addition of arachidonic acid caused significant induction of mPGES-1 and COX-2 in WT cells, whereas COX-1 and cPGES were not affected. Our earlier and the current studies demonstrate the coregulation of cPLA₂, COX, and mPGES-1, in PGE₂ synthesis pathway, and that these enzymes contribute to the elevation of PGE₂ level when one COX isoform is absent.     

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.     

Synthesis of Ethyl Tfa- and Tfp- Amino Acid-p-Aminobenzoates and Their Juvenile Hormone Test

Adipocytes can function as endocrine cells secreting a variety of adipocytokines including tumor necrosis factor (TNF)-α. Treatment of cultured mouse 3T3-L1 preadipocytes with TNF-α induced apoptosis, as was evident from increases in nuclear condensation and caspase-3 activity, but differentiated adipocytes during the maturation phase showed resistance to apoptosis by TNF-α. Antioxidants effectively reduced TNF-α-induced apoptosis in preadipocytes, indicating the involvement of reactive oxygen species. Exposure of preadipocytes to calcium ionophore A23187 reduced TNF-α-induced apoptosis, which was accompanied by increased production of prostaglandins (PGs) E₂ and PGF_2α. TNF-αpreferentially promoted gene expression of cyclooxygenase (COX)-2 without affecting that of COX-1. Consistently, NS-398, a COX-2 inhibitor, stimulated TNF-α-induced apoptosis, which was reversed by exogenous PGE₂ and PGF_2α. These results indicate that endogenous PGE₂ and PGF_2α synthesized by preadipocytes through the induction of COX-2 can serve as anti-apoptotic factors against apoptosis by TNF-α.     

Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE₂) is one of the main catabolic factors involved in OA. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE₂. For both COX and PGES, three isoforms have been described: in cartilage, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. COX-3 (a variant of COX-1) and mPGES-2 have been recently cloned but little is known about their expression and regulation in cartilage, as is also the case for 15-PGDH. We investigated the regulation of the genes encoding COX and PGES isoforms during mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) for 2 to 24 hours. After determination of the amount of PGE₂ released in the media (enzyme immunoassay), mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blotting respectively. Mechanical compression of cartilage explants significantly increased PGE₂ production in a time-dependent manner. This was not due to the synthesis of IL-1, since pretreatment with interleukin 1 receptor antagonist (IL1-Ra) did not alter the PGE₂ synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression, whereas COX-3 and mPGES-2 mRNA expression was not modified. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE₂ synthesis after 18 hours, suggesting that PGE₂ synthesis could be altered by the induction of 15-PGDH expression. We conclude that, along with COX-2, dynamic compression induces mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Synthesis,biological evaluation,and docking analysis of a novel family of 1-methyl-1H-pyrrole-2,5-diones as highly potent and selective cyclooxygenase-2 (COX-2) inhibitors

As a continuous research for discovery of new COX-2 inhibitors, we present the simple chemical synthesis, in vitro biological screening, and molecular docking study of 1H-pyrrole-2,5-dione derivatives. New synthetic compounds were evaluated for the inhibitory activities on LPS-induced PGE₂ production in RAW 264.7 macrophage cells as well as the COX-1 and COX-2 inhibitory potency. Among them, compound 9d (MPO-0029) was identified as more potent and selective COX-2 inhibitor [PGE₂ IC₅₀ = 8.7 nM, COX-2 IC₅₀ = 6.0 nM; COX-2 selectivity index (SI) = >168] than celecoxib. Molecular docking experiments were further performed against COX-2 and COX-1 isozymes to determine their probable binding models. Results of molecular docking studies revealed that compound 9d (MPO-0029) has stronger binding interaction with COX-2 than with COX-1 isozyme, and provided successfully complementary theoretical support for the obtained experimental biological data.     

Suppression of adipogenesis program in cultured preadipocytes transfected stably with cyclooxygenase isoforms

Prostaglandins (PGs) are known to play a variety of roles in adipocytes and precursor cells, which have the arachidonate cyclooxygenase (COX) pathway to generate several series of PGs at different stages of life cycle of adipocytes. To gain a unique insight into the specific roles of the COX isoforms during the life cycle of adipocytes, 3T3-L1 preadipocytes were stably transfected with a mammalian expression vector harboring either cDNA coding for murine COX-1 or COX-2. The cloned stable transfectants with COX-1 or COX-2 exhibited higher expression levels of their corresponding mRNA and proteins, and greater production of PGE₂ upon stimulation with free arachidonic acid or A23187 than the parent cells and the transfectants with vector only. However, either type of transfectants brought about the marked reduction in the accumulation of triacylglycerols after the standard adipogenesis program. Unexpectedly, aspirin or other COX inhibitors at different phases of life cycle of adipocytes failed to reverse the reduced storage of fats. The transfectants with COX-2 were sensitive to exogenous 15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) and troglitazone as peroxisome proliferator-activated receptor γ (PPARγ) agonists during the maturation phase for restoring the adipogenesis. By contrast, the transfectants with COX-1 were much less sensitive, which was reflected by much lower gene expression levels of PPARγ and the related adipocyte-specific markers. Taken together, the results suggest that the sustained overexpression of either COX-1 or COX-2 resulted in the interference of adipogenesis program through a PG-independent mechanism with a different mode of action of COX isoforms.