Original article



Molecular Pharmacology Laboratory Chair of Pharmacology Medical College of Jagiellonian University, Grzegrzecka 16 31-531 Krakow, Poland

  Prostaglandin endoperoxide H2 (PGH2) is generated from arachidonic acid by either constitutive (COX-1) or inducible (COX-2) cyclooxygenases. In arterial wall PGH2 is converted by PGI2 synthase (PGI-S) to prostacyclin (PGI2), and in platelets by thromboxane synthase (TX-S) to thromboxane (TXA2). Other prostanoids as PGD2, PGF2alpha or PGE2 were believed to arise non-enzymatically from PGH2. Only recently, human prostaglandin E synthase (PGE-S) has been identified and cloned as a membrane bound, microsomal, glutathione-dependent inducible enzyme. Here we demonstrated that interleukin 1ß (IL-1ß) is an inducer of COX-2 and PGE-S in human umbilical vein endothelial cells (HUVEC). Functional expression of PGE-S was measured at the level of specific mRNA by semi-quantitative RT-PCR, PGE-S protein was detected by Western blot in HUVEC, while PGE2 was measured by immunoassay in the supernatant. Actinomycin D, a classical transcription inhibitor, was used to prove that indeed IL-1ß induced the functional PGE-S enzyme. PGE2 generation in HUVEC was inhibited by indomethacin, acetamoniphen and dexamethasone. In conclusion, we found that in cultured endothelial cells IL-1ß induced as evidenced by the appearance of its transcript and its functional enzyme. The induction of endothelial PGE-S and COX-2 appeared to be and their transcripts were induced as fast as one might expect from immediate early genes. It means that IL-1ß-triggered-PGE2 biosynthesis in endothelial cells is probably regulated by induction of both COX-2 and PGE-S. This is way we hypothesise the existence of at least two distinct pools of COX-2: the first selectively coupled to PGE-S and the second one that is coupled to PGI-S yielding the main endothelial product - PGI2.

Key words:    prostaglandin E2 synthase, cyclooxygenase, interleukin 1ß, prostaglandin E2, functional gene expression


Prostaglandin endoperoxidase H synthase (EC, PGHS) is a bifunctional enzyme. Firstly it catalyzes the conversion of arachidonic acid to PGG2 via the cyclooxygenase activity, and then PGG2 is converted to prostaglandin endoperoxide H2 (PGH2) via the peroxidase activity. Two PGHS isoenzymes, also named COX-1 and COX-2, were identified and cloned (1-6). COX-derived PGH2 occupies a central position in the biosynthesis of prostanoids. Since 1976 it is known that in arterial wall PGH2 is metabolised by PGI2 synthase (PGI-S) to prostacyclin (7) and in platelets by TXA2 synthase (TX-S) to TXA2 (8). Other prostanoids such as PGD2, PGF2alpha or PGE2 were thought to be generated non- enzematically from PGH2 (9-12). In early eighties, Gerritsen and Printz proposed an unknown enzyme to be responsible for isomerisation of PGH2 to PGE2, but their attempts to purify microsomal PGE isomerase failed (13). Not long ago Jakobsson et al. identified and cloned human prostaglandin E synthase (EC, PGE-S) as a membrane-bound, microsomal, glutathione-dependent, inducible enzyme (14). The second PGE-S isoform in cytosol was suggested (15-17). Cytosolic PGE-S seems to be identical to p23, a chaperon for the hsp90/glucocorticoid receptor complex (17). In many types of cells microsomal PGE-S is induced by cytokines (i.e. interleukin 1ß (IL-1ß) or tumour necrosis factor a (TNFalpha) and it is glucocorticoid repressible (16). Abundant expression of PGE-S was reported in cancer cell line as A549 and HeLa. The PGE-S is also expressed at intermediate levels in prostate, testis, placenta, mammary gland and bladder (14). The presence of PGE-S is moreover reported in several colorectal adenomas (18). Some time ago PGE-S was found in heart and brain (19). In vasculature the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that PGE-S mRNA was present in smooth muscle cells but not in human umbilical vein endothelial cells (HUVEC) and not in endothelial cells from human saphenous vein, even following stimulation with IL-1ß, TNFalpha, phorbol myristate acetate or lipopolysaccharide (11). However, it is known that endothelial cells, along with PGI2, do produce also PGE2, in particular after cytokine stimulation (13;20-22).

Here we report on the mechanism of induction of functional PGE-S in HUVEC using interleukin 1ß as an inducer.


Endothelial cell culture and drug used

Human umbilical vein endothelial cells (HUVEC) were cultured as previously described (23). All experiments were performed on HUVEC at the second passage on reaching confluence. Prior to experiments, the cells were maintained 24 hours in the culture medium (Optimem, Gibco, Invitrogen, UK) without serum. The culture medium was replaced before adding of human recombinant interleukin 1ß (IL-1ß, 0.02-200 pM, R&D Systems, Germany), actinomycin D (5 g ml-1 , Sigma,.USA), dexamethasone (0.3-3 M, Sigma, USA), indomethacin (1-10 M, Sigma, USA) and acetaminophen (30-300 M, Sigma, USA) during various periods of time. Then culture supernants were collected for PGE2 assay while the cells were subjected to RNA extraction or protein analysis.

Reverse-transcription polymerase chain reaction for endothelial enzymes

Total RNA was extracted from HUVEC using TRIZOL Reagent (Invitrogen, Life Technologies, UK). Reverse transcription (RT) of total RNA (1 g) was performed with oligo- (dT)12-18 primer and M-MLV reverse transcriptase (Gibco Invitrogen, UK) during 2 hours at 42C. PCR experiments were performed with 1 l of the first strand DNA (cDNA) in 25 l mixture containing 200 M dNTPs, 1.5 mM MgCl2, 40 nM of specific primer pairs and 1U of recombinant HotStart Taq™ polymerase (Qiagen, USA) using T3 Thermocycler (Biometra, Germany) (24). The sequence of the sense and antisense primers for human PGE-S were: 5'-ctctgcagcacgctgctgg-3' and 5'-gtaggtcacggagcggatgg-3' (11). After an initial denaturation of PCR sample for 15 min at 95C, 34 cycles of amplification (94C for 45 sec, 65C for 45 sec and 72C for 45 sec) were performed followed by 10 min extention at 72C. Specific primer pairs for COX-1 and COX-2 were purchased from Ambion (USA) with the thermocycler profile: an initial denaturation for 15 min at 95C, 23 - 34 cycles of amplification (94C for 30 sec, 59C for 30 sec and 72C for 30 sec) followed by 10 min extention at 72C. The quality of RNA samples was evaluated using ß-actin specific primers: 5'-agcgggaaatcgtgcgtg-3' (sense) and 5'-agcgggaaatcgtgcgtg-3' (antisense). PCR products were detected by electophoresis on 2% agarose gels stained with ethidium bromide. Images were captured electronically with DC40 digital camera (Kodak, USA) and the bands were quantified using image analysis software (NIH Image, Scion Co., USA). Results were normalized and expressed as ratio of pixel density units for specific mRNA to ß-actin mRNA. For position and size of observed specific bands DNA marker M1 (pUC19/Mspl, Gdask, Poland) was run in parallel.

Expression of PGE-S, COX-1 and COX-2 proteins

HUVEC were lysed in phosphate-buffered saline containing 0,1% sodium dodecyl sulphate (SDS) and samples (30 mg of total protein per lane) were applied to SDS-polyacrylamide (12% for COXs and 15% for PGE-S) gels and subdued to electrophoresis using Mini Protean II (Bio-Rad, USA) and Laemmli buffer system (20;25). Then proteins were electroblotted onto nitrocelulose membranes with a semidry blotter (Bio-Rad, USA). The membranes were blocked overnight in 4C with 5% non-fat dried milk. Then the membranes were incubated 2 hrs in room temperature with specific murine antibody to PGE-S(COX-1 or COX-2) protein (Cayman Chemical, USA). Bands were visualized with nitro blue tetrazolium and 5-bromo-4chloro-3-indolyl phosphate as substrates for alkaline phosphatase-conjugated secondary antibody (Sigma, USA) Protein bands were scanned with CD40 digital camera (Kodak, USA).

Assay of PGE2

Culture supernatants (500 l) were collected in Eppendorff tubes and stored at -70C not longer than for a week. PGE2 was assayed using the monoclonal enzyme immunoassay (EIA) kit (Cayman Chemical, USA) according to the manufacturer's protocol. Results were expressed in pg ml-1.

Fig. 1. Induction of COX-2 and PGE-S transcripts by IL-1ß at a range of concentrations 0.02 pM - 200 pM. Panel A - actual gels. M1 - the marker lane, 1 - 6 lanes: RT-PCR products after incubation with IL-1ß at concentrations indicated in the graph. Panel B shows levels of specific mRNA for PGE-S (filled bars) and COX-2 (patterned bars) expression normalized with respect to b-actin in HUVEC and treated with increasing concentrations of


Three hours after the exposition of HUVEC to IL-1ß at concentrations from 0.02 pM to 200 pM, total RNA was extracted and specific mRNA for PGE-S and 645.COX-2 were analysed by RT-PCR. Fig.1A shows data obtained from gels. Fig.1B shows the expression levels of PGE-S mRNA and COX-2 mRNA standardized to ß-actin. A concentration-dependent sharp increase in the expression level of PGE-S (filled bars) and COX-2 (patterned bars) was evoked by IL-1ß at concentrations from 0.02 pM to 2.0 pM. At a higher concentration of IL-1ß no further significant increase in response occurred. To confirm the specificity of RT-PCR products, they were sequenced and then blasted against known sequences in GenBank database. Our specific PGE-S amplified in RT-PCR product had >99% homology to that stored in GenBank (data nor shown). After 3 hours of stimulation of endothelial cells with increasing concentration of IL-1ß, HUVEC were lysed and analysed with Western blot technique. Fig.2 shows the appearance of the band of PGE-S protein in lane four, five and to lesser extent in lane 6. We observed band for COX-2 protein in control, unstimulated endothelial cells. However, the treatment with IL-1ß increased the COX-2 protein expression in a concentration dependent manner. No effect of stimulation with IL-1ß was observed in the case of COX-1 protein expression. Three hours after stimulation of HUVEC with increasing concentration of IL-1ß, we collected supernatants and PGE2 was assayed with EIA. Fig.3A shows an increase in release of PGE2 while the concentration of IL-1ß was rising from 0.02 pM to 2 pM. Then PGE2 release into the culture medium reached its plateau and IL-1ß at higher concentrations no longer augmented the release of PGE2. We also measured the kinetics of PGE2 release into HUVEC supernatants. Cells were stimulated with IL-1ß (2 pM) and samples were collected at time intervals shown on Fig.3B. PGE2 release increased linearly at all time intervals and maximum, almost 20 fold increase in PGE2 release, occurred at 180 min of stimulation with the cytokine. As shown on Fig.4A endothelial cells when pre-treated with actinomycin D (Act-D, 5 g ml-1 ), did not respond with formation of PGE-S mRNA to stimulation with IL-1ß (2 pM). Filled bars (Fig.4B) show normalized expression levels of specific PGE-S mRNA plotted against ß-actin. Similarly, pre-treatment with act-D inhibits expression of COX-2 mRNA (Fig.4A). Patterned bars show normalized against ß-actin level of specific COX-2 mRNA expression (Fig.4B). In contrast, expression level of COX-1 remained unchanged both after act-D pre-treatment and after IL-1ß stimulation (Fig.4). Finally, we performed pharmacological analysis of PGE2 release from HUVEC stimulated 3 hours with IL-1ß (2 pM). Actinomycin D (5 g ml-1 ) prevented IL-1ß-induced PGE2 release into supernatant (see Fig.5). Also dexamethasone (0.3 -3 M), acetaminophen (30- 300 M) and indomethacin (1-10 M) blocked PGE2 release from HUVEC after IL-1ß stimulation (Fig.5).

Fig. 2. PGE-S and COX-2 but not COX-1 protein expression in HUVEC is dependent on IL-1ß concentration as evidenced by Western blot.


Our results indicate clearly that endothelial cells (HUVEC) did express the PGE-S transcript and the PGE-S protein next to the stimulation with IL-1ß. The presence of functional PGE-S in HUVEC was confirmed by semi-quantitative RT-PCR analysis (Fig.1), immunostaining of PGE-S protein (Fig.2) and the rapid release of PGE2 into culture supernatants (Fig.3). Actinomycin D inhibited at the transcriptional level the IL-1ß induced appearance of PGE-S mRNA (Fig.4), and in consequence it inhibited the release of PGE2 into the culture medium (Fig.5). Our above finding differs from the data presented by Soler et al.(11) even though we used the same specific primer pair and RT-PCR profile for PGE-S mRNA analysis. The reason for this discrepancy remains unclear. A number of subcultivations of cultured endothelial cells does influence their PGI2 production (26, 27). Might it influence PGE2 generating system in a similar way? We used HUVEC of the 2nd passage while Soler et al. used endothelial cells of the 1st passage (11).

Fig. 3. Effect of IL-1ß on PGE2 release from HUVEC. Panel A - Concentration dependence (IL-1ß, 0.02 - 200 pM). Panel B - Time-dependence (15-180 min) for IL1ß at a concentration 2 pM.

In line with the report of Jakobsson et al. (14) we used IL-1ß as an inducer for PGE2 generating system. This proinflammatory cytokine might be of relevance in the endothelial cell dysfunction accompanying atherosclerosis (28). Our results (Fig.1-3) combined with known effect of IL-1ß on COX-2 induction also suggest that PGE-S and COX-2 are coregulated and that the biosynthesis of PGE2 may depend on the presence of both of these enzymes. Indeed, an inducible PGE synthase activity that coincides with COX-2 expression was described in lipopolysaccharide-stimulated rat macrophages (29-31). The functional coupling between microsomal COX-2 and PGE-S that leads to the efficient PGE2 production from arachidonate was reported by Murakami et al. (16). In the same paper colocalization of these two enzymes was visualized by confocal microscopic analyses and demonstrated in cell lines cotransfected with COX-2 and PGE-S.

Fig. 4. Panel A - HUVEC when pre-treated with actinomycin D (Act-D, 5g ml-1 ) did not respond with formation of PGE-S mRNA and COX-2 mRNA to stimulation with IL-1ß (2 pM) as evidenced by RT-PCR. The expression level of COX-1 remained unchanged neither to IL-1ß stimulation nor Act-D pre-treatment. Panel B - Bars shows the normalized expression levels of specific PGE-S mRNA, COX-2 mRNA and COX-1 mRNA plotted against ß-actin.

Cytokine-induced COX-2 and PGE2 production is strongly repressed by dexamethasone in a number of experimental systems (32-34). We observed that repression of IL-1ß-induced COX-2 and IL-1ß-induced PGE-S by dexamethasone involved some degree (<30%) of transcriptional repression (data not shown). Newton et al. also observed 25-40% suppression of COX-2 transcription (34). Mechanisms of dexamethasone action on transcriptional repression includes transrepression of glucocorticoid response elements (35), repression of AP-1-dependent transactivation (36), upregulation of NF-B inhibitor (IBalpha) (37-39). However, this is not sufficient to account for the observed profound inhibition at the product level (Fig.5). Additional postranscriptional mechanisms involving loss of poly(A) mRNA are proposed for the total dexamethasone-dependent blockage of PGE2 release (34). The complex mechanisms of the suppression by glucocorticoides of endothelial prostanoids generating systems certainly deserve further studies.

Fig. 5. The effect of acti- nomycin D, acetaminophen, indomethacin and dexame- thasone on IL-1ß (2 pM) - dependent release of PGE2 determined after incubation with IL-1ß alone or in the presence of indicated drugs.

The anti-inflammatory benefits of nonsteroidal anti-inflammatory drugs (non- selective COX inhibitors, NSAIDs) are usually ascribed to inhibition of COX-2, while their undesirable side effects rely on COX-1 inhibition (40). Recent data with selective COX-2 inhibitors (e.g. coxibs) stay in line with the above reasoning. As shown in Fig.5 indomethacin (a NSAID) at concentrations of 1 M and 10 M strongly inhibited PGE2 release. Profound inhibition of PGE2 release from IL-1ß-induced HUVEC by acetaminophen (Fig.5) was rather an unexpected finding. Although acetaminophen is a potent antipyretic and analgesic drug, in vitro it is a weak inhibitor of COX-1 and COX-2. In mouse macrophage cell line, the diclofenac-induced COX-2 was more sensitive to inhibition with acetaminophen than the lipopolysaccharide-induced COX-2 (41;42). This may point out to this particular COX-2 variant with specific subcellular localization, protein modifications or redox state (41;42) as to a possible candidate to COX-3 (43;44). Other possibilities of inhibitory action of acetaminophen might derive from its side effects such as producing mitochondrial dysfunction, peroxynitrite formation or glutathione depletion (45, 46). Since PGE-S is glutathione- dependent inducible enzyme (14), inhibitory activity of acetaminophen may relay not only on the COX-2 inhibition but also on the glutathione depletion. Indeed, in bovine or ram seminal vesicle microsomes we had proved that acetaminophen in presence of glutathione inhibited COX activity, while in absence of glutathione it stimulated COX activity (47).

COX-2 is hardly detected in most mammalian tissues, but its expression can be rapidly induced (2-6 hours) in endothelial cells (48). In this paper we confirmed this fast (3 hours) induction of COX-2 mRNA (Fig.1 and Fig.4) and COX-2 protein (Fig.2). In addition, we observed a rapid induction of PGE-S transcripts (Fig.1 and Fig.4), PGE-S protein (Fig.2) and PGE-S product (Fig.3B). In our model PGE-S behaved as a product of an immediate early gene.

So far, in many cellular systems, induction of COX-2 was associated with shifting from PGI2 towards PGE2 generation (29-31;49). Here is an explanation that we can offer. In endothelial cells along with the induction of COX-2 occurs induction of PGE-S. It may well be that there exists a special pool of COX-2 ("COX-2-bis") selectively coupled with PGE-S. This "COX-2-bis" may show distinct distribution in tissues and cells or may be governed by divergent transcriptional and postranscriptional regulation (41-43). Our data show that unstimulated HUVEC express both COX-2 mRNA (see Fig.1 and Fig.4) and COX-2 protein (Fig.2) but they do not release PGE2 into the culture supernatant (Fig.3). This happens after the treatment with IL-1ß. The above speaks for "COX- 2 bis" hypothesis. One may speculate that in endothelial cells shear stress (50, 51) and bradykinin (52) induce "classical" COX-2 coupled with PGI-S while proinflammatory cytokines induce "COX-2 bis". This assumption certainly needs more studies to be verified.

Acknowledgements: The authors thank Ms. Renata Budzyska for her excellent technical help. These studies were supported by grant 4 P05A 050 19 from the State Committee for Scientific Research and grants 501/P/150/L and W/149/P/L from Medical College of Jagiellonian University.

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R e c e i v e d:  September 17, 2002
A c c e p t e d: October 29, 2002

Authors address: Wojciech Uracz, MD, Ph.D., Molecular Pharmacology Laboratory Chair of Pharmacology Medical College of Jagiellonian University, Grzegorzecka 16 31-531 Krakow, Poland, Tel.: (012) 421 36 23, Fax: (012) 422 25 47
e-mail: miuracz@kinga.cyf-kr.edu.pl