Up-regulation of adipogenesis in adipocytes expressing stably cyclooxygenase-2 in the antisense direction

Adipocytes and the precursor cells express two types of cyclooxygenase (COX) isoforms that are involved in the biosynthesis of different types of prostaglandins (PGs) exerting opposite effects on adipogenesis. To evaluate the role of the inducible COX-2 isoform in the control of the differentiation and maturation of adipocytes, we employed an antisense technology to suppress specifically the expression of COX-2 in adipocytes. Cultured 3T3-L1 preadipocytes were transfected stably with a mammalian expression vector having the full-length cDNA encoding mouse COX-2 oriented in the antisense direction. The cloned transfectants with antisense COX-2 exhibited stable expression of antisense RNA for COX-2, which was accompanied by the suppressed expression of mRNA and protein levels of sense COX-2. However, almost no alteration in the expression of COX-1 was detected. The transfectants with antisense COX-2 showed significant decreases in the delayed synthesis of PGE₂ involving the inducible COX-2 in response to cell stimuli. By contrast, the immediate synthesis of PGE₂ associated with the constitutive COX-1 was not influenced appreciably. The stable expression of antisense mRNA of COX-2 resulted in significant stimulation of fat storage during the maturation phase without affecting the cell proliferation associated with the clonal expansion phase. The gene expression studies revealed higher expression levels of adipocyte-specific markers in the transfectants with antisense COX-2, indicating the mechanism that stimulates adipogenesis program. The up-regulation of fat storage was appreciably prevented by anti-adipogenic prostanoids, such as PGE₂ and PGF_2α, during the maturation phase. These results suggest that COX-2 is more preferentially involved in the generation of endogenous anti-adipogenic prostanoids during the maturation phase of adipocytes.     

Gene expression of isoformic enzymes in arachidonate cyclooxygenase pathway and the regulation by tumor necrosis factor alpha during life cycle of adipocytes

Adipocytes serve not only as a storage depot of fats but also as endocrine cells secreting adipocytokines including tumor necrosis factor alpha (TNFalpha). Using preadipogenic 3T3-L1 cells, we attempt to determine the response of adipocytes at different stages of the life cycle to TNFalpha with respect to the gene expression of the arachidonate cyclooxygenase (COX) pathway and the role of endogenous prostaglandins (PGs). The gene expression analysis of the COX pathway revealed the marked increase in mRNA and protein levels of COX-2 in response to TNFalpha in preadipocytes, whereas COX-1 was expressed constitutively. Moreover, the cells at different cycle stages exhibited the specific gene expression of isoformic enzymes of prostaglandin (PG) synthases for PGs of the D(2), E(2), and F(2alpha) series upon exposure to TNFalpha. The treatment of preadipocytes with TNFalpha along with calcium ionophore A23187 resulted in the stimulated formation of PGE(2) and PGF(2alpha), attenuating the apoptotic cell death induced by TNFalpha alone. The response of adipocytes to synthesize these PGs declined during the differentiation and maturation phases. The cells during the differentiation phase were the most sensitive to TNFalpha in terms of the decrease in adipogenesis without the mediation of endogenous PGs. TNFalpha was also effective in suppressing adipogenesis during the maturation process. Taken together, TNFalpha can control cell number of preadipocytes as well as the size of fat storage in mature adipocytes. The action of TNFalpha on preadipocytes can be modulated by the production of endogenous PGs through the induction of COX-2.     

Gene expression of arachidonate cyclooxygenase pathway leading to the delayed synthesis of prostaglandins E2 and F2alpha in response to phorbol 12-myristate 13-acetate and action of these prostanoids during life cycle of adipocytes

Several types of prostaglandin (PG)s are synthesized in adipocytes and involved differently in the control of adipogenesis. To elucidate how the PG synthesis is regulated at different stages in the life cycle of adipocytes, we examined the gene expression of arachidonate cyclooxygenase (COX) pathway leading to the delayed synthesis of PGE2 and PGF2alpha and their roles in adipogenesis after exposure of cultured cells to phorbol 12-myristate 13-acetate (PMA), which is a useful system for monitoring mitogen-induced changes. While the expression of COX-1 remained constitutive, mRNA and protein levels of COX-2 were up-regulated by treatment with PMA. Preadipocytes exhibited higher gene expression of cytosolic phospholipase A2alpha (cPLA2alpha) and PGF synthase. In contrast, three isoforms of PGE synthase are expressed constitutively during all phases. The delayed synthesis of PGE2 and PGF2alpha following the stimulation for 24 with a mixture of PMA and calcium ionophore A23187 was the highest in preadipocytes, reflecting the increased expression levels of cPLA2alpha and COX-2. Cultured cells treated with PMA during the differentiation phase and then exposed to the maturation medium, or cells treated with PMA in the maturation medium after the differentiation phase showed the suppression of adipogenesis in adipocytes. The attenuating effect of PMA was additionally enhanced when the cell were treated along with A32187 during the differentiation phase, suggesting the involvement of endogenous PGs. The cells at the stages of the differentiation and maturation phases were highly sensitive to exogenous PGE2 and PGF2alpha, respectively, resulting in the marked suppression of the stored fats in adipocytes. Taken together, these results provided the evidence for the distinct gene expression of isoformic enzymes in the COX pathway leading to the synthesis of PGE2 and PGF2alpha and the specific action of these prostanoids at different cycle stages of adipocytes.     

Prostaglandin receptor EP1-mediated differential degradation of cyclooxygenases involves a specific lysine residue

The cyclooxygenase (COX) enzyme isoforms COX-1 and COX-2 catalyze the main step in the generation of prostanoids that mediate major physiological functions. Whereas COX-1 is a ubiquitously expressed stable protein, COX-2 is transiently upregulated in many pathologies and is often associated with a poor prognostic outcome. We have recently shown that an interaction of COX-2 with the prostaglandin EP₁ receptor accelerates its degradation via a mechanism that augments its level of ubiquitination. Here we show that the sensitivity of both COX-1 and COX-2 to EP₁ is altered upon modification of one lysine residue. A point mutation of lysine to-arginine in position 432 of COX-2 (K432R) yields an enzyme with decreased sensitivity to EP₁-mediated degradation. In contrast, insertion of a putative ubiquitination site into the corresponding position of COX-1 (H446K′) yields an enzyme with higher levels of ubiquitination and reduced expression. Furthermore, compared to wild type COX-1, H446K′ is significantly more sensitive to downregulation by EP₁. Together these data suggest that distinctive ubiquitination of COX-1 and COX-2 may be responsible for their different sensitivity to EP₁-mediated degradation.     

COX-derived prostanoid pathways in gastrointestinal cancer development and progression: Novel targets for prevention and intervention

Arachidonic acid metabolism through cyclooxygenase (COX) pathways leads to the generation of biologically active eicosanoids. Eicosanoid expression levels vary during development and progression of gastrointestinal (GI) malignancies.COX-2 is the major COX-isoform responsible for G.I. cancer development/progression. COX-2 expression increases during progression from a normal to cancerous state. Evidence from observational studies has demonstrated that chronic NSAID use reduces the risk of cancer development, while both incidence and risk of death due to G.I. cancers were significantly reduced by daily aspirin intake. A number of randomized controlled trials (APC trial, Prevention of Sporadic Adenomatous Polyps trial, APPROVe trial) have also shown a significant protective effect in patients receiving selective COX-2 inhibitors. However, chronic use of selective COX-2 inhibitors at high doses was associated with increased cardiovascular risk, while NSAIDs have also been associated with increased risk. More recently, downstream effectors of COX-signaling have been investigated in cancer development/progression. PGE₂, which binds to both EP and PPAR receptors, is the major prostanoid implicated in the carcinogenesis of G.I. cancers. The role of TXA₂ in G.I. cancers has also been examined, although further studies are required to uncover its role in carcinogenesis. Other prostanoids investigated include PGD₂ and its metabolite 15d-PGJ2, PGF_1α and PGI₂. Targeting these prostanoids in G.I. cancers has the promise of avoiding cardiovascular toxicity associated with chronic selective COX-2 inhibition, while maintaining anti-tumor reactivity.A progressive sequence from normal to pre-malignant to a malignant state has been identified in G.I. cancers. In this review, we will discuss the role of the COX-derived prostanoids in G.I. cancer development and progression. Targeting these downstream prostanoids for chemoprevention and/or treatment of G.I. cancers will also be discussed. Finally, we will highlight the latest pre-clinical technologies as well as avenues for future investigation in this highly topical research field.     

Chronic Administration of Lamotrigine Downregulates COX-2 mRNA and Protein in Rat Frontal Cortex

Chronic administration to rats of mood-stabilizers that are effective against mania in bipolar disorder, is reported to downregulate markers of the brain arachidonic acid cascade. We hypothesized that chronic administration of lamotrigine, which is used to treat depression and rapid cycling in bipolar disorder, might do so as well. Male CDF rats were administered a therapeutically relevant dose of lamotrigine (10 mg/kg) or vehicle intragastrically once daily for 42 days. Protein levels of isoforms of phospholipase A₂ (PLA₂) and of cyclooxygenase (COX), and the mRNA level of COX-2, were quantified in the frontal cortex using immunoblotting and RT-PCR, respectively. Compared to vehicle-treated rats, chronic lamotrigine significantly decreased frontal cortex protein and mRNA levels of COX-2 without altering protein levels of the PLA₂ isoforms. Consistent with the hypothesis, lamotrigine and other mood-stabilizers have a common downregulatory action on COX-2 expression in rat brain, which may account in part for their efficacy in bipolar disorder.     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

15-Deoxy-Δ-prostaglandin J2 impairs the functions of histone acetyltransferases through their insolubilization in cells

The cyclopentenonic prostaglandin 15-deoxy-Δ^12,14-PG J₂ (15d-PGJ₂) is a metabolite derived from PGD₂. Although 15d-PGJ₂ has been demonstrated to be a potent ligand for peroxisome proliferator activated receptor γ (PPARγ), the functions are not fully understood. In order to examine the effect of 15d-PGJ₂ on histone acetyltransferases (HATs), several lines of cell including mouse embryonic fibroblast (MEF) cells were exposed to 15d-PGJ₂. Three types of HAT, p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF), selectively disappeared from the soluble fraction in time- and dose-dependent manners. Inversely, HATs in the insoluble fraction increased, suggesting their conformational changes. The decrease in the soluble form of HATs resulted in the attenuation of NF-κB-, p53-, and heat shock factor-dependent reporter gene expressions, implying that the insoluble HATs are inactive. The resultant insoluble PCAF and p300 seemed to be digested by proteasome, because proteasome inhibitors caused the accumulation of insoluble HATs. Taken together, these results indicate that 15d-PGJ₂ attenuates some gene expressions that require HATs. This inhibitory action of 15d-PGJ₂ on the function of HATs was independent of PPARγ, because PPARγ agonists could not mimick 15d-PGJ₂ and PPARγ antagonists did not inhibit 15d-PGJ₂.     

Time-dependent changes in the brain arachidonic acid cascade during cuprizone-induced demyelination and remyelination

Phospholipases A₂ (PLA₂) are the enzymatic keys for the activation of the arachidonic acid (AA) cascade and the subsequent synthesis of pro-inflammatory prostanoids (prostaglandins and tromboxanes). Prostanoids play critical roles in the initiation and modulation of inflammation and their levels have been reported increased in several neurological and neurodegenerative disorders, including multiple sclerosis (MS).Here, we aimed to determine whether brain expression PLA₂ enzymes and the terminal prostagland in levels are changed during cuprizone-induced demyelination and in the subsequent remyelination phase.Mice were given the neurotoxicant cuprizone through the diet for six weeks to induce brain demyelination. Then, cuprizone was withdrawn and mice were returned to a normal diet for 6 weeks to allow spontaneous remyelination.We found that after 4-6 weeks of cuprizone, sPLA₂(V) and cPLA₂, but not iPLA₂(VI), gene expression was upregulated in the cortex, concomitant with an increase in the expression of astrocyte and microglia markers. Cyclooxygenase (COX)-2 gene expression was consistently upregulated during all the demyelination period, whereas COX-1 sporadically increased only at week 5 of cuprizone exposure. However, we found that at the protein level only sPLA₂(V) and COX-1 were elevated during demyelination, with COX-1 selectively expressed by activated and infiltrated microglia/macrophages and astrocytes. Levels of PGE₂, PGD₂, PGI₂ and TXB₂ were also increased during demyelination. During remyelination, none of the PLA₂ isoforms was significantly changed, whereas COX-1 and -2 were sporadically upregulated only at the gene expression level. PGE₂, PGI₂ and PGD₂ levels returned to normal, whereas TXB₂ was still upregulated after 3 weeks of cuprizone withdrawal.Our study characterizes for the first time time-dependent changes in the AA metabolic pathway during cuprizone-induced demyelination and the subsequent remyelination and suggests that sPLA₂(V) is the major isoform contributing to AA release.     

Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes

Prostacyclin alternatively called prostaglandin (PG) I₂ is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI₂ has not been determined comprehensively at different life stages of adipocytes. PGI₂ is rapidly hydrolyzed to the stable product, 6-keto-PGF_1α, in biological fluids. Therefore, the generation of PGI₂ can be quantified as the amount of 6-keto-PGF_1α. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF_1α. According to the typical calibration curve of our ELISA, 6-keto-PGF_1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF_2α. The resulting ELISA was applied to the quantification of 6-keto-PGF_1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF_1α during the maturation phase of 4–6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI₂. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF_1α is useful for monitoring the endogenous levels of the unstable parent PGI₂ at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.     

Effects of protein kinase C activation on prostaglandin production and cyclooxygenase mRNA levels in ovine astroglia

We examined effects of protein kinase C (PKC) activation by phorbol dibutyrate (PDB) on prostaglandin production in astroglia. Astroglia were cultured from sheep fetal cortex and grown in Eagle's basal media supplemented with 10% fetal calf serum (BME-C). Prostaglandin F_2a (PGF_2α) levels in media were determined at 2–24 hours after exposure to PDB. PDB increased production of PGF_2α at 10⁻⁸M and 10⁻⁶M. In addition, PDB increased the ratio of membrane to cytosolic PKC. Coapplication of H7 [1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine] (10⁻⁴M) with PDB (10⁻⁶M) inhibited PDB-induced PGF_2a production. To investigate the role of protein synthesis in increased prostaglandin production by PDB, astroglia were coincubated with actinomycin D (1 mg/ml) or cycloheximide (10 mg/ml). At 4 hrs, both actinomycin D and cycloheximide inhibited increases in PGF_2a in response to PDB application. In addition, COX-2 mRNA levels and COX activity levels were examined. PDB increased COX-2 mRNA levels by 2 hours, and COX activity tripled after 12 hr exposure to PDB. In addition, the increase in COX activity was blocked by cycloheximide. In summary, PKC activation promotes enhanced prostaglandin production via an increase in COX synthesis.     

Inhibitory effect of hot-water extract of quince (<Emphasis Type="Italic">Cydonia oblonga</Emphasis>) on immunoglobulin E-dependent late-phase immune reactions of mast cells

We evaluated the effect of a crude hot-water extract (HW) of quince (Cydonia oblonga Miller) fruit on immunoglobulin E (IgE)-dependent late-phase immune reactions of mast cells using in vitro system. Mast cell-like RBL-2H3 cells were treated with quince HW and late-phase reaction was then induced by stimulation with IgE + Antigen. Quince HW reduced the elevation of interleukin-13 and tumor necrosis factor-α expression level. Furthermore, quince HW suppressed these cytokine expressions of mouse bone marrow-derived mast cells (BMMCs), a normal mast cell model. Leukotriene C₄ and prostaglandin D₂ production in BMMCs after 1 and 6 h of stimulation, respectively, were also reduced by treating the cells with quince HW. We found that the induction of intracellular cyclooxygenase (COX)-2 expression but not COX-1 expression in BMMCs was reduced by quince HW. These results suggest that quince HW has an inhibitory effect on broad range of the late-phase immune reactions of mast cells.