A cyclooxygenase-2/prostaglandin E2 pathway augments activation-induced cytosine deaminase expression within replicating human B cells

Within inflammatory environments, B cells encountering foreign or self-Ag can develop tertiary lymphoid tissue expressing activation-induced cytosine deaminase (AID). Recently, this DNA-modifying enzyme was detected in nonlymphoid cells within several inflamed tissues and strongly implicated in malignant transformation. This study examines whether a cyclooxygenase 2 (COX-2) pathway, often linked to inflammation, influences AID expression in activated B lymphocytes. In this paper, we report that dividing human B cells responding to surrogate C3d-coated Ag, IL-4, and BAFF express AID, as well as COX-2. A progressive increase in AID with each division was paralleled by a division-related increase in a COX-2-linked enzyme, microsomal PGE(2) synthase-1, and the PGE(2)R, EP2. Cells with the greatest expression of AID expressed the highest levels of EP2. Although COX-2 inhibitors diminished both AID expression and IgG class switching, exogenous PGE(2) and butaprost, a selective EP2 agonist, augmented AID mRNA/protein and increased the numbers of IgG(+) progeny. Despite the latter, the proportion of IgG(+) cells within viable progeny generally declined with PGE(2) supplementation. This was not due to PGE(2)-promoted differentiation to plasma cells or to greater downstream switching. Rather, because phosphorylated ataxia telangiectasia mutated levels were increased in progeny of PGE(2)-supplemented cultures, it appears more likely that PGE(2) facilitates AID-dependent DNA double-strand breaks that block B cell cycle progression or promote activation-induced cell death, or both. Taken together, the results suggest that a PGE(2) feed-forward mechanism for augmenting COX-2 pathway proteins promotes progressively increased levels of AID mRNA, protein, and function.     

Stimulation of prostaglandin E2 and thromboxane B2 production by human monocytes in response to interleukin-2

Interleukin 2 (IL-2) is a potent lymphokine involved in the regulation of immune responses and is classically regarded as a stimulus for the activation and growth of T-cells. Recent reports have demonstrated the IL-2 dependent activation of human peripheral blood lymphocytes into lymphokine activated killer cells capable of lysing tumor cells both in vitro and in vivo. In this study we report data which clearly show IL-2 may also act to down-regulate the immune response by inducing the synthesis of arachidonic acid metabolites with known immunosuppressive actions. Stimulation of peripheral human blood monocytes with IL-2 caused an increased production of prostaglandin E2 (PGE2) and thromboxane (TXB2) in a dose-dependent manner. Kinetic analysis showed no increase above controls after 6 hours and maximal levels by 10 hours; elevated levels were maintained after 45 hours of incubation. After 20 hours of stimulation with 2000 U/ml IL-2, the level of PGE2 and TXB2 were greater than three-fold above controls, 0.7 and 19 ng/10(6) cells, respectively. The stimulation was relatively specific in that neither prostacyclin nor leukotrienes were produced in response to IL-2. These data demonstrate that IL-2 acts on human monocytes to induce the secretion of PGE2 and TXB2.     

Evidence for a role of phosphodiesterase 4 in lipopolysaccharide-stimulated prostaglandin E2 production and matrix metalloproteinase-9 activity in human amniochorionic membranes

Chorioamniotic infection is a leading cause of preterm premature rupture of fetal membranes (amnion and chorion). Bacterial infection induces an inflammatory response characterized by elevated production of proinflammatory cytokines; the latter activate the production of both PGs that stimulate uterine contractions, and matrix metalloproteinases (MMPs) that degrade the extracellular matrix of the chorioamniotic membranes. The inflammatory response is under the control of cAMP content, which is partly regulated by phosphodiesterases (PDE). In this study, we investigated the role of the PDE4 family in the inflammatory process triggered by LPS in a model of amniochorionic explants. We found that PDE4 family is the major cAMP-PDE expressed in human fetal membranes and that PDE4 activity is increased by LPS treatment. Selective inhibition of PDE4 activity affected LPS signaling, because PDE4 inhibitors (rolipram and/or cilomilast) reduced the release of the proinflammatory cytokine TNF-alpha and increased the release of the anti-inflammatory cytokine IL-10. PDE4 inhibition reduced cyclooxygenase-2 protein expression and PGE(2) production and also modulated MMP-9, a key mediator of the membrane rupture process, by inhibiting pro-MMP-9 mRNA expression and pro-MMP-9 activity. These results demonstrate that the PDE4 family participates in the regulation of the inflammatory response associated with fetal membrane rupture during infection. The PDE4 family may be an appropriate pharmacological target for the management of infection-induced preterm delivery.     

Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human gingival fibroblasts

Interleukin-1 (IL-1) plays a crucial role in the immunopathological responses involved with tissue destruction in chronic inflammatory diseases, such as periodontal disease, as it stimulates host cells including fibroblasts to produce various inflammatory mediators and catabolic factors. We comprehensively investigated the involvement of mitogen-activated protein kinases (MAPKs)/activator protein-1 (AP-1) and IkappaB kinases (IKKs)/IkappaBs/nuclear factor-kappaB (NF-kappaB) in IL-1beta-stimulated IL-6, IL-8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase-1 (MMP-1) production by human gingival fibroblasts (HGF). Three MAPKs, extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK), which were simultaneously activated by IL-1beta, mediated subsequent c-fos and c-jun mRNA expression and DNA binding of AP-1 at different magnitudes. IKKalpha/beta/IkappaB-alpha/NF-kappaB was also involved in the IL-1 signaling cascade. Further, IL-1beta stimulated HGF to produce IL-6, IL-8, PGE(2) and MMP-1 via activation of the 3 MAPKs and NF-kappaB, as inhibitors of each MAPK and NF-kappaB significantly suppressed the production of IL-1beta-stimulated factors, though these pathways might also play distinct roles in IL-1beta activities. Our results strongly suggest that the MAPKs/AP-1 and IKK/IkappaB/NF-kappaB cascades cooperatively mediate the IL-1beta-stimulated synthesis of IL-6, IL-8, PGE(2) and MMP-1 in HGF.     

Autocrine/paracrine prostaglandin E2 production by non-small cell lung cancer cells regulates matrix metalloproteinase-2 and CD44 in cyclooxygenase-2-dependent invasion

Tumor cyclooxygenase-2 (COX-2) expression is known to be associated with enhanced tumor invasiveness. In the present study, we evaluated the importance of the COX-2 product prostaglandin E2 (PGE2) and its signaling through the EP4 receptor in mediating non-small cell lung cancer (NSCLC) invasiveness. Genetic inhibition of tumor COX-2 led to diminished matrix metalloproteinase (MMP)-2, CD44, and EP4 receptor expression and invasion. Treatment of NSCLC cells with exogenous 16,16-dimethylprostaglandin E2 significantly increased EP4 receptor, CD44, and MMP-2 expression and matrigel invasion. In contrast, anti-PGE2 decreased EP4 receptor, CD44, and MMP-2 expression in NSCLC cells. EP4 receptor signaling was found to be central to this process, because antisense oligonucleotide-mediated inhibition of tumor cell EP4 receptors significantly decreased CD44 expression. In addition, agents that increased intracellular cAMP, as is typical of EP4 receptor signaling, markedly increased CD44 expression. Moreover, MMP-2-AS treatment decreased PGE2-mediated CD44 expression, and CD44-AS treatment decreased MMP-2 expression. Thus, PGE2-mediated effects through EP4 required the parallel induction of both CD44 and MMP-2 expression because genetic inhibition of either MMP-2 or CD44 expression effectively blocked PGE2-mediated invasion in NSCLC. These findings indicate that PGE2 regulates COX-2-dependent, CD44- and MMP-2-mediated invasion in NSCLC in an autocrine/paracrine manner via EP receptor signaling. Thus, blocking PGE2 production or activity by genetic or pharmacological interventions may prove to be beneficial in chemoprevention or treatment of NSCLC.     

Inhibition of phospholipase activity in human monocytes by IFN-gamma blocks endogenous prostaglandin E2-dependent collagenase production

Previous studies from our laboratory have demonstrated that exposure of human monocytes to a stimulant, such as Con A, results in the production of the enzyme collagenase through PGE2-dependent pathway. Inasmuch as rIFN-gamma has been shown to modulate monocyte/macrophage PG synthesis, we examined the effect of rIFN-gamma on the activation sequence leading to collagenase production. The addition of rIFN-gamma (10 to 1000 U/ml) to Con A-stimulated monocytes resulted in a dose-dependent inhibition of PGE2 and collagenase synthesis. The suppression of collagenase production by rIFN-gamma was related to its ability to reduce PGE2 levels as demonstrated by the restoration of collagenase activity by the addition of PGE2. HPLC analysis of the arachidonic acid (AA) metabolites released by monocytes showed that rIFN-gamma caused a reduction in the release of AA and products of the cyclooxygenase and lipoxygenase pathways. These data indicated that rIFN-gamma decreased eicosanoid production by inhibiting the release of AA from phospholipids. This conclusion was supported by the reduction in membrane bound phospholipase activity in rIFN-gamma-treated monocytes. Moreover, the inhibition by rIFN-gamma of PGE2 and collagenase was reversed by the addition of phospholipase A2. Our findings demonstrate that rIFN-gamma inhibits phospholipase activity in activated monocytes and as a result blocks PGE2-dependent collagenase synthesis.     

Exogenous prostaglandin E2 inhibits TPA induced matrix metalloproteinase-9 production in MCF-7 cells

Elevated levels of prostaglandin E2 (PGE2) have been reported in many high metastatic human breast cancers, but no relationship between exogenous PGE2 activity, expression of matrix metalloproteinases (MMPs) and metastasis in human tumor cells has been reported. The poorly invasive human breast cancer cell line MCF-7 was cultured for 24h in the presence of both phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 nM) and PGE2 (1 microM) and the activity of MMP-9, one of the MMPs involved in metastasis, was measured, in growth medium by gelatin substrate zymography. TPA induced a strong production of MMP-9 while exogenous PGE2 had no effect on the basal MMP-9 level, but inhibited the TPA induced enzyme expression and matrigel invasiveness. We showed that MCF-7 cells expressed EP2, EP3 and EP4 receptors for PGE2 and that its action was probably mediated by EP4 receptor and adenylyl cyclase activation while cAMP dependent PKA was not involved in the process of inhibition of MMP-9 production. These findings suggest a possible inhibitory role for exogenous PGE2 in the metastatic process development.     

Role of glucose in interleukin-1β production by lipopolysaccharide-activated human monocytes

When monocytes are activated with endotoxin (lipopolysaccharide [LPS]), they make and release several mediators, including interleukin-1β (IL-1β). This study was undertaken to investigate the role of glucose in IL-1β production by these cells. IL-1β was produced in a dose-dependent manner to glucose concentration in the culture medium. The uptake of (³H)2-deoxyglucose in monocytes was stimulated by LPS 1,554% after 10 minutes, 6,095% after 2 hours, then gradually declined after 4 hours of incubation. The inhibition of the uptake of (³H)2-deoxyglucose by either 10 μM cytochaiasin B or phloretin, added at the time of monocyte activation, was accompanied by significant reduction in ATP/ADP ratio and the inhibition of the production of IL-1β by activated monocytes. The synthesis of total protein did not change in monocytes activated in the absence of glucose in the culture medium, nor in the presence of either 10 μM cytochalasin B or phloretin. The export of IL-1β from LPS-activated monocytes was not inhibited by either 10 μM cytochalasin B or phloretin, nor in the absence of glucose in the culture medium. These data suggest that (1) glucose is required for LPS-induced IL-1β production by monocytes; (2) glucose is the major source of ATP for IL-1β production; (3) glucose transporter (GLUT 1) does not control the export of IL-1β. © 1993 Wiley-Liss, Inc.     

Human cytomegalovirus induced cyclooxygenase-2 in human retinal pigment epithelial cells augments viral replication through a prostaglandin pathway

Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kappaB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p<0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

Mannose-containing molecular patterns are strong inducers of cyclooxygenase-2 expression and prostaglandin E2 production in human macrophages

The induction of cyclooxygenase-2 (COX-2) and the production of PGE(2) in response to pathogen-associated molecular patterns decorated with mannose moieties were studied in human monocytes and monocyte-derived macrophages (MDM). Saccharomyces cerevisiae mannan was a robust agonist, suggesting the involvement of the mannose receptor (MR). MR expression increased along the macrophage differentiation route, as judged from both its surface display assessed by flow cytometry and the ability of MDM to ingest mannosylated BSA. Treatment with mannose-BSA, a weak agonist of the MR containing a lower ratio of attached sugar compared with pure polysaccharides, before the addition of mannan inhibited COX-2 expression, whereas this was not observed when agonists other than mannan and zymosan were used. HeLa cells, which were found to express MR mRNA, showed a significant induction of COX-2 expression upon mannan challenge. Conversely, mannan did not induce COX-2 expression in HEK293 cells, which express the mRNA encoding Endo180, a parent receptor pertaining to the MR family, but not the MR itself. These data indicate that mannan is a strong inducer of COX-2 expression in human MDM, most likely by acting through the MR route. Because COX-2 products can be both proinflammatory and immunomodulatory, these results disclose a signaling route triggered by mannose-decorated pathogen-associated molecular patterns, which can be involved in both the response to pathogens and the maintenance of homeostasis.     

Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells

Substance P (SP) via its neurokinin-1 receptor (NK-1R) regulates several gastrointestinal functions. We previously reported that NK-1R-mediated chloride secretion in the colon involves formation of PG. PGE2 biosynthesis is controlled by cyclooxygenase-1 (COX-1) and COX-2, whose induction involves the STATs. In this study, we examined whether SP stimulates PGE2 production and COX-2 expression in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and identified the pathways involved in this response. SP exposure time and dose dependently induced an early (1-min) phosphorylation of JAK2, STAT3, and STAT5, followed by COX-2 expression and PGE2 production by 2 h. Pharmacologic experiments showed that PGE2 production is dependent on newly synthesized COX-2, but COX-1 protein. Inhibition of protein kinase Ctheta (PKCtheta), but not PKCepsilon and PKCdelta, significantly reduced SP-induced COX-2 up-regulation, and JAK2, STAT3, and STAT5 phosphorylation. Pharmacological blockade of JAK inhibited SP-induced JAK2, STAT3, and STAT5 phosphorylation; COX-2 expression; and PGE2 production. Transient transfection with JAK2 short-interferring RNA reduced COX-2 promoter activity and JAK2 phosphorylation, while RNA interference of STAT isoforms showed that STAT5 predominantly mediates SP-induced COX-2 promoter activity. Site-directed mutation of STAT binding sites on the COX-2 promoter completely abolished COX-2 promoter activity. Lastly, COX-2 expression was elevated in colon of mice during experimental colitis, and this effect was normalized by administration of the NK-1R antagonist CJ-12,255. Our results demonstrate that SP stimulates COX-2 expression and PGE2 production in human colonocytes via activation of the JAK2-STAT3/5 pathway.     

Phagocytosis of hemozoin enhances matrix metalloproteinase-9 activity and TNF-alpha production in human monocytes: role of matrix metalloproteinases in the pathogenesis of falciparum malaria

Matrix metalloproteinase-9 (MMP-9), secreted by activated monocytes, degrades matrix proteins, disrupts basal lamina, and activates TNF-alpha from its precursors. In turn, TNF-alpha enhances synthesis of MMP-9 in monocytes. We show here that trophozoite-parasitized RBCs/hemozoin-fed adherent human monocytes displayed increased MMP-9 activity and protein/mRNA expression, produced TNF-alpha time-dependently, and showed higher matrix invasion ability. MMP-9 activation was specific for trophozoite/hemozoin-fed monocytes, was dependent on TNF-alpha production, and abrogated by anti-TNF-alpha Ab and by a specific inhibitor of MMP-9/MMP-13 activity. Hemozoin-induced enhancement of MMP-9 and TNF-alpha production would have a 2-fold effect: to start and feed a cyclic reinforcement loop in which hemozoin enhances production of TNF-alpha, which in turn induces both activation of MMP-9 and shedding of TNF-alpha into the extracellular compartment; and, second, to disrupt the basal lamina of endothelia. Excess production of TNF-alpha and disruption of the basal lamina with extravasation of blood cells into perivascular tissues are hallmarks of severe malaria. Pharmacological inhibition of MMP-9 may offer a new chance to control pathogenic mechanisms in malaria.     

Oxidative stress and the development of diabetic retinopathy: Contributory role of matrix metalloproteinase-2

Matrix metalloproteinases (MMPs) degrade extracellular matrix and regulate many functions including cell signaling. Oxidative stress is implicated in the development of diabetic retinopathy, and MMP-2, the most ubiquitous member of the MMP family, is sensitive to oxidative stress. This study aimed to determine the regulation of MMP-2 by oxidative stress in the development of diabetic retinopathy and the role of MMP-2 in the apoptosis of retinal capillary cells. The effects of mitochondrial superoxide scavenger on glucose-induced alterations in MMP-2, and its proenzyme activator MT1-MMP and physiological inhibitor TIMP-2, were determined in retinal endothelial cells, and the regulation of their glucose-induced accelerated apoptosis by the inhibitors of MMP-2 was accessed. To confirm in vitro results, the effects of antioxidant supplementation on MMP-2, MT1-MMP, and TIMP-2 were investigated in the retina of streptozotocin-induced diabetic rats. Glucose-induced activation of retinal capillary cell MMP-2 and MT1-MMP and decrease in TIMP-2 were inhibited by superoxide scavengers, and their accelerated apoptosis was prevented by the inhibitors of MMP-2. Antioxidant therapies, which have been shown to inhibit oxidative stress, capillary cell apoptosis, and retinopathy in diabetic rats, ameliorated alterations in retinal MMP-2 and its regulators. Thus, MMP-2 has a proapoptotic role in the loss of retinal capillary cells in diabetes, and the activation of MMP-2 is under the control of superoxide. This suggests a possible use of MMP-2-targeted therapy to inhibit the development of diabetic retinopathy.     

Interleukin-1beta induces interleukin-6 production through the production of prostaglandin E(2) in human osteoblasts, MG-63 cells.

This study was conducted to investigate the mechanism of interleukin-1beta (IL-1beta)-induced IL-6 production in human osteoblasts (MG-63 cells). Stimulation with IL-1beta resulted in the production of IL-6 and prostaglandin E(2) (PGE(2)). IL-6 production gradually increased and peaked 96 h after stimulation. IL-6 mRNA was detected between 4 and 72 h after IL-1beta stimulation. The patterns of PGE(2) production and the expression of cyclooxygenase-2 (COX-2) mRNA were biphasic after stimulation. Actinomycin D, cycloheximide, indomethacin, and NS-398 (COX-2 inhibitor) suppressed the production of IL-6 and PGE(2). Anti-PGE(2) antibody markedly reduced the production of IL-6. In addition, stimulation with 17-phenyl-PGE(2), a PGE receptor-1 (EP-1 receptor) agonist, led to the expression of IL-6 mRNA after pretreatment with IL-1beta. These findings indicate that IL-1beta-induced IL-6 production in MG-63 cells involves the following sequence of steps: IL-1beta-induced COX-2 activation, PGE(2) production, and EP-1 receptor signaling prior to IL-6 production.     

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.     

Protein kinase Cdelta-mediated CREB activation regulates ghrelin-induced cyclooxygenase-2 expression and prostaglandin E2 production in human colonic epithelial cells

Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone release and appetite. Our recent study showed that ghrelin could stimulate protein kinase C-mediated activation of nuclear factor-kappaB (NF-kappaB) and interleukin-8 secretion in human colonic epithelial cells transfected with a functional ghrelin receptor. In the present study, the effect of ghrelin stimulation on cyclooxygenese-2 expression and prostaglandin E2 production was examined. The data indicate that ghrelin significantly increased the levels of cyclooxygenase-2 (COX-2) protein as well as its promoter activity, which leaded to profound increase in prostaglandin E2 secretion. In order to examine the involvement of NF-kappaB and cAMP responsive element-binding protein (CREB) in this response, the NF-kappaB inhibitory protein IkappaBalpha or a dominant negative mutant of CREB was co-transfected into cells and the data show that transfection of either IkappaBalpha or DN-CREB significantly attenuated ghrelin-induced COX-2 expression. Moreover ghrelin stimulated phosphorylation of CREB, which was mediated primarily via protein kinase Cdelta activation. Furthermore, inhibition of PKCdelta function significantly attenuated ghrelin-induced COX-2 expression. In addition, ghrelin stimulates phosphorylation of PKCdelta. Together, these results indicate that in addition to NF-kappaB, protein kinase Cdelta-mediated CREB activation plays an important role in the cellular responses of ghrelin.