Human microvascular endothelial cell prostaglandin E1 synthesis during in vitro ischemia-reperfusion

Ischemia-reperfusion injury is a microvascular event documented in numerous in vivo animal models. In animal models, prostaglandin and prostaglandin analogues have been found to ameliorate reperfusion injury. These studies were undertaken to evaluate human microvascular endothelial PGE(1) synthesis during in vitro ischemia followed by reperfusion. Human (neonatal) microvascular endothelial cell (MEC) cultures (n = 6) were subjected to sequential 2 h periods of normoxia (20% O(2)), ischemia (1.5% O(2)), and reperfusion (20% O(2)). Prostaglandin E(2) synthesis in conditioned media was determined by ELISA. Steady state levels of MEC prostaglandin H synthase (PGHS)-1 and -2 mRNA were assessed at the end of each 2-h period using RT-PCR and a quantitative mRNA ELISA. MEC PGHS protein levels were analyzed using an ELISA. PGE(1) release increased significantly during the initial 30 min of ischemia, but rapidly fell below normoxic levels by 90 and 120 min. During reperfusion, PGE(1) release returned to normoxic levels at 30, 60, and 90 min, and exceeded normoxic levels at 120 min. PGHS-1 mRNA levels were undetectable during all experimental conditions. PGHS-2 mRNA levels were unchanged by ischemia, but were decreased by reperfusion. In contrast, PGHS-2 protein levels increased 3-fold during ischemia, and remained elevated during reperfusion. Human MEC do not express PGHS-1 mRNA in vitro. Prolonged ischemia decreases MEC PGE(1) synthesis, and stimulates increased PGHS-2 protein levels without altering the steady state levels of COX-2 mRNA. During reperfusion, increased PGHS-2 protein levels persist and are associated with stimulated PGE(2) secretion, despite relative decreases in PGHS-2 mRNA.     

Regulation of prostaglandin H synthase activity in dog urothelial cells.

TPA regulation of prostaglandin H synthase activity in primary and subcultured dog urothelial cells was investigated. Previous studies have demonstrated an early (0-2 hr) increase in PGE2 synthesis mediated by TPA which is dependent upon release of endogenous arachidonic acid by a phospholipase-mediated pathway. In this study, prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 microM) to media. PGE2 synthesis, measured by radioimmunoassay, served as an index of prostaglandin H synthase activity. After a 24-hr incubation with 0.1 microM TPA or 1.0 microM A23187, arachidonic acid elicited significantly more PGE2 synthesis in agonist-treated cells than it did in control cells in primary culture. Microsomes from 24-hr TPA-treated cells exhibited significantly more prostaglandin H synthase activity than did those from control cells. In addition, the PGE2 content of overnight media was approximately 10-fold greater in TPA-treated cells than in control cells. The late (24 hr) response was more sensitive to lower concentrations of TPA than was the earlier (0-2 hr) response. TPA at 0.1 microM was a maximum effective dose for both responses. The 24-hr response was blocked by cycloheximide and staurosporine, inhibitors of protein synthesis and protein kinase C, respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late TPA-mediated increase in PGE2 synthesis. Subcultured cells exhibited both an early and a late TPA response. Only the early response was inhibited by aspirin pretreatment. Results suggest that the late response with TPA is caused by de novo synthesis of prostaglandin H synthase. Thus, primary and subcultured dog urothelial cells possess two distinct mechanisms for regulating signal transduction by arachidonic acid metabolism. This study provides a basis for assessing these mechanisms of signal transduction in urothelial cell lines and transformed cells.     

Dexamethasone stimulates arachidonic acid conversion to prostaglandin E2 in human amnion cells

The purpose of this investigation was to study the mechanism of stimulation of PGE2 output from human amnion epithelial cells by the synthetic glucocorticoid dexamethasone. Cells incubated in serum-free pseudo-amniotic fluid produced very low levels of PGE2, even when arachidonic acid (1 microM) was present. Pretreatment of cells with dexamethasone (50 nM) for 21 h increased the PGE2 output 6- to 7-fold in 2-h incubations only in the presence of arachidonic acid. The RNA synthesis inhibitor, actinomycin D (1 microgram/ml), and the protein synthesis inhibitor, cycloheximide (40 micrograms/ml), each blocked dexamethasone-stimulated arachidonic acid conversion to PGE2. The time course of these events suggests that dexamethasone first initiates RNA synthesis. Acetylsalicylic acid, a specific and irreversible blocker of prostaglandin endoperoxide H synthase (cyclooxygenase), was used to determine whether dexamethasone could stimulate new enzyme synthesis. Cells treated first with acetylsalicylic acid (30 min) then dexamethasone (22 h) produced as much PGE2 in response to 1 microM arachidonate as did cells exposed to dexamethasone only. Exposing cells to acetylsalicylic acid after dexamethasone completely eliminated PGE2 output. These data suggest that dexamethasone stimulates the synthesis of prostaglandin endoperoxide H synthase.     

Osteoprotegerin levels increased by interleukin-1beta in human periodontal ligament cells are suppressed through prostaglandin E(2) synthesized de novo

Osteoprotegerin (OPG) is a novel tumor necrosis factor receptor superfamily that inhibits osteoclast differentiation, activity, and survival. Interleukin-1beta (IL-1beta) increases OPG expression. IL-1beta also increases prostaglandin E(2) (PGE(2)) production and stimulates bone resorption. In the present study, we examined the involvement of PGE(2) in IL-1beta-induced increases in OPG levels in human periodontal ligament cells (HPL cells) in an effort to clarify apparently conflicting IL-1beta actions on bone resorption and understand IL-1beta-induced increases in secretion of OPG and PGE(2) in HPL cells. 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, a mRNA synthesis inhibitor, partly inhibited the increase in OPG mRNA levels induced by IL-1beta. Cycloheximide, a protein synthesis inhibitor, enhanced the stimulatory effect of IL-1beta. Etodolac, a selective cyclooxygenase-2 inhibitor, suppressed the increase in PGE(2) levels. Furthermore, etodolac reinforced the promotion of OPG expression by IL-1beta at the mRNA and protein levels. PGE(2) added to cultures of HPL cells decreased OPG mRNA levels in a dose- and time- dependent manner. These findings suggest that the increase in OPG levels induced by IL-1beta in HPL cells is suppressed through PGE(2) synthesized de novo.     

Mechanical stimulation of skeletal muscle cells mitigates glucocorticoid-induced decreases in prostaglandin production and prostaglandin synthase activity

The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content in tissue cultured, avlan skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the effects of Dex and mechanical stretch on prostaglandin production and prostaglandin H synthase (PGHS) activity were examined. In static cultures, 10^?8 M Dex reduced PGF_2α production 55–65% and PGE₂ production 84–90% after 24–72 h of incubation. Repetitive 10% stretch-relaxations of non-Dex-treated cultures increased PGF_2α efflux 41% at 24 h and 276% at 72 h, and increased PGE₂ production 51% at 24 h and 236% at 72 h. Mechanical stimulation of Dex-treated cultures increased PGF_2α production 162% after 24 h, returning PGF_2α efflux to the level of non-Dex-treated cultures. At 72 h, stretch increased PGF_2α efflux 65% in Dex-treated cultures. Mechanical stimulation of Dex-treated cultures also increased PGE₂ production at 24 h, but not at 72 h. Dex reduced PGHS activity in the muscle cultures by 70% after 8–24 h of incubation, and mechanical stimulation of the Dex-treated cultures increased PGHS activity by 98% after 24 h. Repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by mitigating the Dex-induced declines in PGHS activity and prostaglandin production. © 1994 wiley-Liss, Inc.     

Platelet-activating factor stimulates production of prostaglandin E2 in murine osteoblast-like cell line MC3T3-E1.

We found that platelet-activating factor (PAF) stimulated the production of prostaglandin (PG) E2 in MC3T3-E1 cells in a time- and dose-dependent manner. 1.0 microM PAF gave a maximal stimulation of PGE2 production by MC3T3-E1 cells after a 4 hr PAF-treatment. Furthermore, the PAF-induced PGE2 production was abolished by the pre-treatment of the cells with a PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine, which occupied the same receptor site as PAF. These results suggest that PAF stimulates the PGE2 synthesis through a PAF receptor mediated pathway. Possibly PAF modulates bone metabolism by stimulating PGE2 synthesis.     

Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis.

Prostaglandins and NO. are important mediators of inflammation and other physiological and pathophysiological processes. Continuous production of these molecules in chronic inflammatory conditions has been linked to development of autoimmune disorders, coronary artery disease, and cancer. There is mounting evidence for a biological relationship between prostanoid biosynthesis and NO. biosynthesis. Upon stimulation, many cells express high levels of nitric oxide synthase (NOS) and prostaglandin endoperoxide synthase (PGHS). There are reports of stimulation of prostaglandin biosynthesis in these cells by direct interaction between NO. and PGHS, but this is not universally observed. Clarification of the role of NO. in PGHS catalysis has been attempted by examining NO. interactions with purified PGHS, including binding to its heme prosthetic group, cysteines, and tyrosyl radicals. However, a clear picture of the mechanism of PGHS stimulation by NO. has not yet emerged. Available studies suggest that NO. may only be a precursor to the molecule that interacts with PGHS. Peroxynitrite (from O2.-+NO.) reacts directly with PGHS to activate prostaglandin synthesis. Furthermore, removal of O2.- from RAW 267.4 cells that produce NO. and PGHS inhibits prostaglandin biosynthesis to the same extent as NOS inhibitors. This interaction between reactive nitrogen species and PGHS may provide new approaches to the control of inflammation in acute and chronic settings.     

Cytoplasmic prostaglandin E2 synthase is dominantly expressed in cultured KAT-50 thyrocytes,cells that express constitutive prostaglandin-endoperoxide H synthase-2. Basis for low protaglandin E2 production

The recent identification and cloning of two glutathione-dependent prostaglandin E(2) synthase (PGES) genes has yielded important insights into the terminal step of PGE(2) synthesis. These enzymes form efficient functional pairs with specific members of the prostaglandin-endoperoxide H synthase (PGHS) family. Microsomal PGES (mPGES) is inducible and works more efficiently with PGHS-2, the inflammatory cyclooxygenase, while the cytoplasmic isoform (cPGES) pairs functionally with PGHS-1, the cyclooxygenase that ordinarily exhibits constitutive expression. KAT-50, a well differentiated thyroid epithelial cell line, expresses high levels of PGHS-2 but surprisingly low levels of PGE(2) when compared with human orbital fibroblasts. Moreover, PGHS-1 protein cannot be detected in KAT-50. We report here that KAT-50 cells express high basal levels of cPGES but mPGES mRNA and protein are undetectable. Thus, KAT-50 cells express the inefficient PGHS-2/cPGES pair, and this results in modest PGE(2) production. The high levels of cPGES and the absence of mPGES expression result from dramatic differences in the activities of their respective gene promoters. When mPGES is expressed in KAT-50 by transiently transfecting the cells, PGE(2) production is up-regulated substantially. These observations indicate that naturally occurring cells can express a suboptimal profile of PGHS and PGES isoforms, resulting in diminished levels of PGE(2) generation.     

Fibroblast growth factor upregulates PGG/H synthase in rabbit microvascular endothelial cells by a glucocorticoid independent mechanism.

The present study was undertaken to determine the effects of acidic fibroblast growth factor (aFGF) on eicosanoid synthesis in microvessel endothelial cells derived from rabbit left ventricular muscle (RCME cells). We observed that aFGF increased AA conversion to PGE2 in a time- and dose-dependent manner, and the stimulatory effect was abolished by actinomycin D and cycloheximide. Acidic FGF increased the recovery of PGG/H synthase activity following aspirin treatment, suggesting an action on de novo PGG/H synthase synthesis. Acidic FGF increased the incorporation of [35S] methionine into a 70 kD immunoreactive PGG/H synthase band. PGG/H synthase synthesis following aspirin treatment was also increased by transforming growth factor beta, while epidermal growth factor basic FGF and platelet derived growth factor were without effect. In addition, the actions of aFGF on de novo PGG/H synthase were compared in several endothelial preparations. Acidic FGF treatment of aspirin treated endothelial cells from rabbit lung microvessels and small pulmonary artery and from human lung microvessels all showed an increase in PGG/H synthase recovery. In contrast, similar treatment of human umbilical vein endothelial cells was without effect. Pretreatment of RCME cells with dexamethasone (1 microM) did not alter the aFGF induction of PGG/H synthase activity. We conclude that aFGF stimulates PGE2 production by a mechanism that includes the de novo synthesis of PGG/H synthase. This mechanism appears to be distinct from previously described glucocorticoid sensitive translational controls of PG synthase synthesis by epidermal growth factor in smooth muscle and mesangial cells.     

Mechanism of the elevation in cardiolipin during HeLa cell entry into the S-phase of the human cell cycle

CL (cardiolipin) is a key phospholipid involved in ATP generation. Since progression through the cell cycle requires ATP we examined regulation of CL synthesis during S-phase in human cells and investigated whether CL or CL synthesis was required to support nucleotide synthesis in S-phase. HeLa cells were made quiescent by serum depletion for 24 h. Serum addition resulted in substantial stimulation of [methyl-(3)H]thymidine incorporation into cells compared with serum-starved cells by 8 h, confirming entry into the S-phase. CL mass was unaltered at 8 h, but increased 2-fold by 16 h post-serum addition compared with serum-starved cells. The reason for the increase in CL mass upon entry into S-phase was an increase in activity and expression of CL de novo biosynthetic and remodelling enzymes and this paralleled the increase in mitochondrial mass. CL de novo biosynthesis from D-[U-(14)C]glucose was elevated, and from [1,3-(3)H]glycerol reduced, upon serum addition to quiescent cells compared with controls and this was a result of differences in the selection of precursor pools at the level of uptake. Triascin C treatment inhibited CL synthesis from [1-(14)C]oleate but did not affect [methyl-(3)H]thymidine incorporation into HeLa cells upon serum addition to serum-starved cells. Barth Syndrome lymphoblasts, which exhibit reduced CL, showed similar [methyl-(3)H]thymidine incorporation into cells upon serum addition to serum-starved cells compared with cells from normal aged-matched controls. The results indicate that CL de novo biosynthesis is up-regulated via elevated activity and expression of CL biosynthetic genes and this accounted for the doubling of CL seen during S-phase; however, normal de novo CL biosynthesis or CL itself is not essential to support nucleotide synthesis during entry into S-phase of the human cell cycle.     

Autocrine/paracrine regulation of apoptosis in epithelial cells by prostaglandin E2

This study was designed to investigate the effect of IL-1alpha-induced up-regulation of cyclooxygenase-2 (COX-2) on prostaglandin E(2) (PGE(2)) secretion and the subsequent phenotypic effects of PGE(2) on epithelial cells. The effect of IL-1alpha on COX-2 expression was investigated in the T24 bladder epithelial cell line following treatment with 0, 0.05, 0.5, 1 or 10 ng/ml IL-1alpha for 1, 2, 4 or 6 h. Quantitative PCR confirmed up-regulation of expression of COX-2 with maximal expression observed following treatment with 0.5 ng/ml IL-1alpha for 1 h. Co-treatment of the cells with 0.5 ng/ml IL-1alpha in the presence or absence of 100 ng/ml IL-1 receptor antagonist (RA) abolished the up-regulation in COX-2 expression confirming that the effect of IL-1alpha is mediated via its membrane-bound receptors. Treatment with 0.5 ng/ml IL-1alpha resulted in a time-dependent increase in PGE(2) secretion with maximal secretion detected at 24 and 48 h after stimulation with IL-1alpha. Co-treatment of the cells with IL-1alpha and IL-1RA or the COX-2 enzyme inhibitor NS398 abolished the IL-1alpha mediated secretion of PGE(2). Treatment of T24 cells with 100 nM PGE(2) resulted in a significant elevation in cAMP generation confirming the expression of functional PGE(2) receptors. Finally, the effect of exogenous treatment with PGE(2) on apoptosis of T24 cells was assessed using cell death detection ELISA. T24 cells were treated with camptothecin to induce apoptosis in the presence or absence of 50 or 100 nM PGE(2) or 10 microM forskolin. Treatment of T24 cells with increasing doses of camptothecin alone resulted in a significant increase in the induction of apoptosis (P<0.01). However, co-treatment of the cells with 50 or 100 nM PGE(2) or 10 microM forskolin resulted in the inhibition of induction of the apoptotic pathway by camptothecin. These data demonstrate that PGE(2) inhibits apoptosis of epithelial cells possibly via cAMP-dependent pathway.     

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1beta-stimulated rat chondrocytes: evidence for PPARgamma-independent inhibition by 15-deoxy-Delta12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.     

Tyrosine kinase inhibitors block the glucocorticoid stimulation of prostaglandin endoperoxide H synthase expression in amnion cells.

Human amnion cells in primary culture respond to glucocorticoids in a characteristic fashion by the increased expression of the inducible prostaglandin endoperoxide H synthase isoenzyme, PGHS-2. Since PGHS-2 induction by agonists generally involves tyrosine kinases, we examined the possibility that the glucocorticoid stimulation of PGHS-2 in the amnion cells is tyrosine kinase dependent. PGHS-2 expression was stimulated in confluent, serum-starved amnion cells with dexamethasone, and the effect of the tyrosine kinase inhibitors herbimycin A and tyrphostins AG126, AG1288, and A1 on enzyme activity induction was determined. All four inhibitors blocked the increase of PGHS activity in a concentration-dependent manner with IC50 values of 0.077 +/- 0.05, 15.38 +/- 5.14, 20.91 +/- 3.1, and 29.77 +/- 8.21 microM, respectively (mean +/- SE, n = 4). Dexamethasone increased (approximately twofold) the tyrosine phosphorylation of 120-, 110-, and 77-kDa proteins in cell extracts, and herbimycin A selectively blocked the phosphorylation of the 110-kDa phosphoprotein. The stimulation of the steady-state level of PGHS-2 mRNA by dexamethasone was also inhibited by herbimycin A. These results suggest that glucocorticoids induce PGHS-2 expression in amnion cells with the involvement of tyrosine kinase(s). The role of tyrosine kinase dependent mechanisms in the control of amnion cell responsiveness to corticosteroids remains to be established.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Role of prostaglandin E2 in the synthesis of the pro-inflammatory cytokine interleukin-6 in primary sensory neurons: an in vivo and in vitro study

Following various types of nerve injury, cyclooxygenase 2 and prostaglandin E2 (PGE2) are universally and chronically up-regulated in injured nerves and contribute to the genesis of neuropathic pain. Persistent high levels of PGE2 likely exert chronic effects on nociceptive dorsal root ganglion (DRG) neurons. In the present study, we tested the hypothesis that injured nerve-derived PGE2 contributes to the up-regulation of the pro-inflammatory cytokine interleukin-6 (IL-6) in DRG neurons following partial sciatic nerve ligation. In naive adult rats, IL-6 was expressed in only a few small size DRG neurons which all co-expressed EP4 receptors. Partial sciatic nerve ligation increased and shifted IL-6 expression from small to medium and large size damaged DRG neurons. Perineural injection of a selective cyclooxygenase 2 inhibitor or a selective EP4 receptor antagonist significantly suppressed the up-regulation of IL-6 in DRG, suggesting that injured nerve derived PGE2 contributes to the de novo synthesis of IL-6 in DRG neurons through EP4 receptors. In cultured sensory ganglion explants, a stabilized PGE2 analog increased IL-6 mRNA and protein levels through the activation of EP4, protein kinase A, protein kinase C, extracellular regulated protein kinase/MAPK, cAMP response element binding protein and NFκB signalling pathways. Taken together, these data indicate that facilitating the de novo synthesis of pain-related cytokines in injured medium and large size DRG neurons is a novel mechanism underlying the role of injured nerve derived PGE2 in the genesis of neuropathic pain.