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.     

Prostaglandin E(2) production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line,RAW 264.7, by Mallotus japonicus phloroglucinol derivatives

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E(2) production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE(2) production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC(50) of 1.0 microM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE(2) production by activated macrophages.     

Prostaglandin H synthase: protein synthesis-independent regulation in bovine aortic endothelial cells

The objective ofthe present study was to examine whether prostaglandin H synthase(PGHS) can be regulated by pathways independent of de novo synthesis ofPGHS. Incubation of bovine aortic endothelial cells (BAEC) for as shortas 5 min with NaF (40 mM) resulted in a 60% increase in PGHS activity.PGHS activity induced by NaF was unaffected by either 10 µMcycloheximide or 1 µM actinomycin D. Aspirin (25 µM) completelyinhibited resting PGHS activity, and NaF did not induce furtherstimulation. NS-398 (500 nM), a specific PGHS-2 inhibitor, wasineffective. Basic fibroblast growth factor (bFGF) induced asignificant increase in PGHS activity within 30 min and was insensitiveto cycloheximide. The levels of PGHS-1 and PGHS-2 proteins, as measuredby Western blots, were not affected by NaF or bFGF. The tyrosine kinaseinhibitor genistein attenuated PGHS activity that was induced by NaFand bFGF, whereas the tyrosine phosphatase inhibitor, sodiumorthovanadate, augmented these responses. The G protein activators5'-guanylyl imidodiphosphate and guanosine5'-O-(3-thiotriphosphate) inhibited both resting andNaF-induced PGHS activities. These results suggest that, in BAEC,PGHS-1 activity can be regulated by tyrosine kinase and/or Gproteins, independently of de novo protein synthesis.

     

Prostaglandin endoperoxide H synthase expression in human thyroid epithelial cells

KAT-50, an established human thyrocyte cell line, expresses constitutively high levels of prostaglandin endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Here, we examine primary human thyrocytes. We find that they, too, express PGHS-2 mRNA and protein under control culture conditions. A substantial fraction of the basal prostaglandin E(2) (PGE(2)) produced by these cells can be inhibited by SC-58125 (5 microM), a PGHS-2-selective inhibitor. Interleukin (IL)-1beta (10 ng/ml) induces PGHS-2 expression and PGE(2) production in primary thyrocytes. The induction of PGHS-2 and PGE(2) synthesis by IL-1beta could be blocked by glucocorticoid treatment. Unlike KAT-50, most of the culture strains also express PGHS-1 protein. Our observations suggest that both cyclooxygenase isoforms may have functional roles in primary human thyroid epithelial cells, and PGHS-2 might predominate under basal and cytokine-activated culture conditions.     

Role of prostaglandin H synthase isoforms in murine ear edema induced by phorbol ester application on skin.

Topical application of TPA to a murine ear induced an edema that was accompanied by eicosanoid biosynthesis and an early enhancement of prostaglandin H synthase 2 (PGHS-2) expression. PGHS-2 induction may be correlated with the time-course of TPA-induced edema formation. Treatment with drugs that inhibit AA mobilization such as dexamethasone or manoalide or inhibitors of leukotriene formation such as zileuton or baicalein, reduced TPA-induced edema development and PGHS-2 levels. On the other hand, arachidonic acid (AA) application on the murine ear induced rapid expression of PGHS-2. This effect was not reproduced by other fatty acids such as oleic, linoleic, eicosatetraynoic or eicosapentaenoic acids. PGHS-2 expression induced by AA application was independent of PGHS and lipoxygenase metabolite synthesis. However, topical application of PGE2 on skin induced PGHS-2 overexpression. This study suggests that AA release and/or subsequent metabolism by PGHS may be involved in the induction of PGHS-2 expression in murine TPA- and AA-induced ear oedema.     

Effects of indomethacin, NS-398 (a selective prostaglandin H synthase-2 inhibitor) and protein synthesis inhibitors on prostaglandin production by the guinea-pig placenta

The outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)were similar from the day 22 guinea-pig placenta and sub-placenta in culture, except for PGE2 output from the sub-placenta which was lower. Between days 22 and 29 of pregnancy, the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)during the initial 2 h culture period increased 6.9-, 1.1- and 3.2-fold, respectively, from the placenta, and 2.1-, 1.4- and 2.2-fold, respectively, from the sub-placenta. Therefore, there was a relatively specific increase in PGF(2 alpha)production by the guinea-pig placenta between days 22 and 29 of pregnancy. The output of PGFM from the cultured placenta also increased between days 22 and 29, indicating that the increase in PGF(2 alpha)output was due to increased synthesis rather than to decreased metabolism. By comparing the amounts of prostaglandins produced by tissue homogenates during a 1 h incubation period, it appears that there is approximately a 2-fold increase in the amount of prostaglandin H synthase (PGHS) present in the guinea-pig placenta between days 22 and 29. NS-398 (a specific inhibitor of PGHS-2) and indomethacin (an inhibitor of both PGHS-1 and PGHS-2) both inhibited prostaglandin production by homogenates of day 22 and day 29 placenta. Indomethacin was more effective than NS-398, except for their actions on PGF(2 alpha)production by the day 29 placenta where indomethacin and NS-398 were equiactive. Indomethacin and NS-398 were both very effective at inhibiting the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)from the day 22 and day 29 placenta and sub-placenta in culture, indicating that prostaglandin production by the guinea-pig placenta and sub-placenta in culture is largely dependent upon the activity of PGHS-2. The high production of PGF(2 alpha)by the day 29 placenta is not dependent on the continual synthesis of fresh protein(s), as inhibitors of protein synthesis did not reduce PGF(2 alpha)output from the day 29 guinea-pig placenta in culture.     

Prostaglandin-endoperoxide H synthase-2 expression in human thyroid epithelium. Evidence for constitutive expression in vivo and in cultured KAT-50 cells.

Prostaglandin-endoperoxide H synthase (PGHS) (EC 1.14.99.1) expression was examined in human thyroid tissue and in KAT-50, a well differentiated human thyroid epithelial cell line. PGHS-1 is found constitutively expressed in most healthy tissues, whereas PGHS-2 is highly inducible and currently thought to be expressed, with few exceptions, only in diseased tissues. Surprisingly, PGHS-2 mRNA and protein were easily detected in normal thyroid tissue. KAT-50 cells express high levels of constitutive PGHS-2 mRNA and protein under basal culture conditions. Compounds usually associated with PGHS-2 induction, including interleukin-1beta (IL-1beta), phorbol 12-myristate 13-acetate, and serum transiently down-regulated PGHS-2 expression. Human PGHS-2 promoter constructs (-1840/+123 and -831/+123) fused to a luciferase reporter and transfected into untreated KAT-50 cells exhibited substantial activity. NS-398, a highly selective inhibitor of PGHS-2 could inhibit substantial basal prostaglandin E2 production. Exogenous IL-1 receptor antagonist or IL-1alpha neutralizing antibodies could attenuate constitutive PGHS-2 expression in KAT-50 cells, suggesting that endogenous IL-1alpha synthesis was driving PGHS-2 expression. Our findings suggest that normal thyroid epithelium expresses high constitutive levels of PGHS-2 in situ and in vitro and this enzyme is active in the generation of prostaglandin E2. Thus, unprovoked PGHS-2 expression might be considerably more widespread in healthy tissues than is currently believed.     

Differential stimulation of prostaglandin G/H synthase-2 in osteocytes and other osteogenic cells by pulsating fluid flow

Mechanical stress produces flow of fluid in the osteocytic lacunar-canalicular network, which is likely the physiological signal for the adaptive response of bone. We compared the induction of prostaglandin G/H synthase-2 (PGHS-2) by pulsating fluid flow (PFF) and serum in osteocytes, osteoblasts, and periosteal fibroblasts, isolated from 18-day-old fetal chicken calvariae. A serum-deprived mixed population of primarily osteocytes and osteoblasts responded to serum with a two- to threefold induction of PGHS-2 mRNA. Serum stimulated PGHS-2-derived PGE(2) release from osteoblasts and osteocytes but not from periosteal fibroblasts as NS-398, a PGHS-2 blocker, inhibited PGE(2) release from osteocytes and osteoblasts with 65%, but not that from periosteal fibroblasts. On the other hand PFF (0.7 Pa, 5 Hz) stimulated (3 fold) PGHS-2 mRNA only in OCY. The related PGE(2) response could be completely inhibited by NS-398. We conclude that osteocytes have a higher intrinsic sensitivity for loading-derived fluid flow than osteoblasts or periosteal fibroblasts.     

Constitutive expression of prostaglandin endoperoxide G/H synthetase (PGHS)-2 but not PGHS-1 in hum an tracheal epithelial cells in vitro

Primary cultures of human tracheal epithelial (HTE) cells cultured in vitro, in defined serum-free media, express prostaglandin endoperoxide G/H synthase (PGHS) activity and produce prostaglandin E₂ (PGE₂). In contrast to every other cell type studied to date, HTE cells appear to constitutively express PGHS-2, the ‘inducible’ form of the enzyme, while expressing little or no PGHS-1, the ‘housekeeping’ isoenzyme in vitro. Prostaglandin synthesis in HTE cells was reduced by a selective PGHS-2 inhibitor, N-(2-cyclohexyloyl-4-nitrophenyl] methane-sulfonamide (NS398), with an IC₅₀ of approximately 1 μM. Immunoblotting and immunoprecipitation of enzymatic activity with isozyme-specific antisera revealed only the PGHS-2 isoform. Full length human cDNA probes detected only PGHS-2 message in Northern blots. Neither PGHS-2 activity nor mRNA levels were dependent on, nor stimulated by peptide growth factors present in the defined serum-free growth medium, or by serum. Prolonged maintenance in the absence of retinoic acid, however, lead to a decline in PGHS activity. Phorbol-myristate acetate (PMA) induced PGHS-2 activity and mRNA and neither PMA-induced, nor constitutive PGHS-2 expression was suppressed by corticosteroids. Actinomycin D-treatment for six hours reduced the PGHS-2 activity and mRNA to only 50% that of untreated cells, suggesting that PGHS-2 mRNA is extremely stable in these cells. HTE cells, at least in vitro, appear unique among prostaglandin-producing cells in that they express PGHS-2, constitutively, independent of regulation by growth factors, serum, or corticosteroids and fail to express PGHS-1 under any culture condition studied.     

Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells

The subcellular colocalization of prostacyclin synthase (PGIS) with prostaglandin H synthase (PGHS) has not been delineated. To test the hypothesis that its colocalization with PGHS is crucial for prostacyclin synthesis, we determined subcellular locations of PGIS, PGHS-1, and PGHS-2 in bovine aortic endothelial cells by immunofluorescent confocal microscopy. PGIS and PGHS-1 were colocalized to nuclear envelope (NE) and endoplasmic reticulum (ER) in resting and adenovirus-infected bovine aortic endothelial cells. PGIS and PGHS-2 were also colocalized to ER in serum-treated or adenovirus-cyclooxygenase-2-infected cells. By contrast, PGIS was not colocalized with PGHS-2 in cells induced with phorbol 12-myristate 13-acetate where PGHS-2 was visualized primarily in vesicle-like structures. The lack of colocalization was accompanied by failed prostacyclin production. Resting ECV304 cells did not produce prostacyclin and had no detectable PGHS-1 and PGIS proteins. Confocal analysis showed abnormal colocalization of PGIS and PGHS-1 to a filamentous structure. Interestingly, the abundant PGIS and PGHS-1 expressed in adenovirus-infected ECV304 cells were colocalized to NE and ER, which synthesized a large quantity of prostacyclin. These findings underscore the importance of colocalization of PGHS and PGIS to ER and NE in prostacyclin synthesis.     

Treatment with prostaglandin F2alpha increases expression of prostaglandin synthase-2 in the rat corpus luteum

Recent studies indicate that the corpus luteum (CL) may be a source of prostaglandin F2alpha (PGF2alpha) for regression. We investigated expression of mRNA and protein for prostaglandin G/H synthase (PGHS) in the CL of immature superovulated rats following administration of PGF2alpha. We observed an increase in mRNA for PGHS-2, the induced isoform, at 1 h and protein at 8 and 24 h after treatment. One hour after PGF2alpha, there was also a progressive decrease in plasma progesterone concentration. There were no changes, however, in expression of PGHS-1, the constitutive isoform, over the 24 h sampling period. These results indicate that PGHS-2 increases following PGF2alpha treatment and that expression of this enzyme in the rat CL may contribute to the luteolytic mechanism.     

Expression of prostaglandin H synthase-1 and -2 in murine intrauterine and gestational tissues from mid pregnancy until term

These studies were undertaken to evaluate the changes in mRNA expression of prostaglandin H synthase (PGHS)-1 and -2 in murine gestational tissues during the latter half of pregnancy. Gestational tissues (decidual caps, membranes surrounding the fetus, and placentae), uterus, and cervix were collected from pregnant mice at days 12, 14, 16, 18, and 19 (am and pm) of gestation (n = 4), and total RNA was isolated and evaluated for PGHS-1 and PGHS-2 expression by northern blot analysis. Expression was normalized to GAPDH. There were no significant increases in PGHS-2 mRNA expression in any of the tissues studied through gestation. In contrast, expression of PGHS-1 mRNA increased significantly at term in the uterus and fetal membranes. In the placenta, mRNA for PGHS-1 was elevated at day 18 and remained elevated over the remainder of the study. These findings suggest that, in the mouse, increased production of PGs by uterine and intrauterine tissues during pregnancy is associated with up-regulation of PGHS-1 and not PGHS-2.     

A computational protocol for the integration of the monotopic protein prostaglandin H2 synthase into a phospholipid bilayer

Prostaglandin H2 synthase (PGHS) synthesizes PGH2, a prostaglandin precursor, from arachidonic acid and was the first monotopic enzyme to have its structure experimentally determined. Both isozymes of PGHS are inhibited by nonsteroidal antiinflammatory drugs, an important class of drugs that are the primary means of relieving pain and inflammation. Selectively inhibiting the second isozyme, PGHS-2, minimizes the gastrointestinal side-effects. This had been achieved by the new PGHS-2 selective NSAIDs (i.e., COX-2 inhibitors) but it has been recently suggested that they suffer from additional side-effects. The design of these drugs only made use of static structures from x-ray crystallographic experiments. Investigating the dynamics of both PGHS-1 and PGHS-2 using classical molecular dynamics is expected to generate new insight into the differences in behavior between the isozymes, and therefore may allow improved PGHS-2 selective inhibitors to be designed. We describe a molecular dynamics protocol that integrates PGHS monomers into phospholipid bilayers, thereby producing in silico atomistic models of the PGHS system. Our protocol exploits the vacuum created beneath the protein when several lipids are removed from the top leaflet of the bilayer. The protein integrates into the bilayer during the first 5 ns in a repeatable process. The integrated PGHS monomer is stable and forms multiple hydrogen bonds between the phosphate groups of the lipids and conserved basic residues (Arg, Lys) on the protein. These interactions stabilize the system and are similar to interactions observed for transmembrane proteins.     

MyoD and Regulation of Prostaglandin H Synthase

MM14 myoblasts, in contrast to their differentiation defective variant (DD-1) cells, do not synthesize detectable levels of prostaglandins or of the initial enzyme in the pathway of prostaglandin synthesis, prostaglandin H synthase (PGHS) but do exhibit readily detectable level of PGHS mRNA (Steiner, S., et al., 1991, Exp. Cell Res. 192, 643). These findings suggest a possible relationship between the myogenic phenotype and the synthesis of prostaglandins. This relationship was examined in the current study by analysis of the effect of transfection of DD-1 cells with a MyoD expression vector (termed MyoDD-1 cells) on expression of MyoD and synthesis of prostaglandins. Proliferating MyoDD-1 cells express readily detectable levels of MyoD protein and mRNA and exhibit markedly diminished levels of PGHS protein and prostaglandins. In contrast, serum-deprived MyoDD-1 cells express little MyoD mRNA or protein and exhibit a readily detectable level of PGHS protein despite having only a slightly higher PGHS mRNA abundance compared to growing MyoDD-1 cells. These studies are consistent with the hypothesis that MyoD expression contributes to the inhibition of prostaglandin synthesis.     

Prostanoid production in Saccharomyces cerevisiae provides a novel assay for nonsteroidal anti-inflammatory drugs

Prostanoids are a large family of lipid mediators originating from prostaglandin H synthase (PGHS) activity on the 20-carbon polyunsaturated fatty acids dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid. The two mouse PGHS isoforms, PGHS-1 and PGHS-2, were expressed in Saccharomyces cerevisiae (yeast), as was a signal-peptide-deleted version of PGHS-1 (PGHS-1MA). PGHS-1 showed high activity with both AA and DGLA as substrate, whereas PGHS-2 activity was high with DGLA but low with AA. Signal peptide removal reduced the activity of PGHS-1MA by >50% relative to PGHS-1, but the residual activity indicated that correct targeting to the lumen of the endoplasmic reticulum may not be necessary for enzyme function. Coexpression of PGHS-1 with cDNAs encoding mouse prostaglandin I synthase and thromboxane A synthase, and with Trypanosoma brucei genomic DNA encoding prostaglandin F synthase in AA-supplemented yeast cultures resulted in production of the corresponding prostanoids, prostaglandin I₂, thromboxane A₂ and prostaglandin F_2α. The inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on prostanoid production were tested on yeast cells expressing PGHS-1 in AA-supplemented culture. Dose-dependent inhibition of prostaglandin H₂ production by aspirin, ibuprofen and indomethacin demonstrated the potential utility of this simple expression system in screening for novel NSAIDs.