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Am J Physiol Heart Circ Physiol 276: H1369-H1378, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 4, H1369-H1378, April 1999

COX-2 and cytosolic PLA2 mediate IL-1beta -induced cAMP production in human vascular smooth muscle cells

Debbie Beasley

Department of Medicine and Tupper Research Institute, New England Medical Center Hospitals, Tufts University School of Medicine, Boston, Massachusetts 02111


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Interleukin (IL)-1 is a potent vasodilator that causes prolonged induction of prostacyclin (PGI2) and cAMP synthesis in human vascular smooth muscle cells (HVSMC). The present study investigated IL-1 induction of PG synthetic enzymes in HVSMC and tested their respective roles in PGI2 and cAMP production. Cyclooxygenase (COX)-1 mRNA was not detectable in either control or IL-1-treated HVSMC, as assessed by RT-PCR. In contrast, COX-2 mRNA was detectable in control HVSMC, increased markedly (16-fold) after 1 h of IL-1 exposure, and increased further (52-fold) after 24 h. COX-2 protein levels, assessed by Western analysis, were increased concomitantly. HVSMC contained mRNA encoding both the secreted and cytosolic forms of phospholipase A2 (sPLA2 and cPLA2, respectively). IL-1 stimulation did not affect sPLA2 mRNA levels, but cPLA2 mRNA levels increased at 8 h, after the initial induction of PG synthesis. HVSMC constitutively expressed PGI2 synthase mRNA, and its levels were not affected by IL-1. A selective COX-2 inhibitor, NS-398, reversed IL-1-induced PGI2 and cAMP production, supporting a role of COX-2 in mediating increased PG synthesis. IL-1-induced cAMP was also reversed by a selective cPLA2 inhibitor, AACOCF3, but not by thioetheramide phosphorylcholine, which inhibits sPLA2 preferentially over cPLA2, supporting a requirement for cPLA2-derived arachidonic acid in IL-1-induced PG synthesis. The delayed induction of cPLA2 mRNA was also attenuated by NS-398, suggesting that it was secondary to the initial COX-2-induced PG synthesis. Together, the results support the hypothesis that IL-1 induces intracellular PG synthesis in HVSMC via rapid upregulation of COX-2, which utilizes cPLA2-derived arachidonic acid to generate PG metabolites that regulate adenylate cyclase.

cyclooxygenase-1; secreted phospholipase A2; prostacyclin synthase; adenylate cyclase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE PROINFLAMMATORY CYTOKINE interleukin-1 (IL-1) is a potent vasodilator, which may contribute to vasodilatation that occurs locally in inflammation and systemically in septic shock. The vasodilatory effect of IL-1 is independent of the endothelium in the blood vessels that have been studied to date and, in different blood vessels, is mediated by either nitric oxide (5, 13) or prostanoids (32, 51). In vascular smooth muscle cells (VSMC) isolated from human blood vessels, induction of prostanoid synthesis is a predominant effect of IL-1 (1, 27). IL-1 induces marked and prolonged increases in prostacyclin (PGI2) synthesis in VSMC derived from human saphenous vein (HVSMC), which in turn results in prolonged activation of adenylate cyclase and increases in the cellular levels of cAMP (6).

Evidence thus far indicates that prostanoid production is regulated at two rate-limiting steps: release of arachidonic acid from membrane phospholipids by phospholipase A2 (PLA2) and conversion of arachidonic acid to the common prostanoid precursor (PGH2) by cyclooxygenase. Two distinct classes of PLA2 enzymes have been proposed to release arachidonic acid from cellular membranes: 14-kDa secretory PLA2 (sPLA2) and an 85-kDa intracellular enzyme, cytosolic PLA2 (cPLA2). Likewise, two forms of cyclooxygenase (COX) can generate PGH2 from arachidonic acid: COX-1, which is constitutively expressed in many cells, and COX-2, which is inducible by a variety of agonists. A predominant effect of IL-1 in many cell types is the rapid upregulation of COX-2 mRNA and protein, with increasd cyclooxygenase activity (42), whereas the levels of COX-1 mRNA remain unchanged. In some cell types, IL-1 also induces expression of either one or both PLA2s. IL-1 induces expression of sPLA2 in rat aortic SMC (37), rat astrocytes (40), rabbit chondrocytes (23), and human hepatoma cells (11); induces expression of cPLA2 in human fibroblasts (20, 29); and induces both PLA2 enzymes in rat mesangial cells (24, 38). In addition, IL-1 may selectively stimulate PGI2 production, because IL-1 has been shown to upregulate PGI2 synthase mRNA in human aortic endothelial cells (34).

The present study investigated the role of prostanoid synthetic enzymes, including distinct forms of COX and PLA2, in IL-1-induced PGI2 and cAMP production in HVSMC. The results provide evidence of an intracellular pathway within HVSMC in which cPLA2-generated arachidonate is converted to prostanoid precursor by COX-2, which in turn results in enhanced prostanoid synthesis and activation of adenylate cyclase. IL-1 activates this pathway in a protein synthesis-dependent fashion, primarily via rapid upregulation of COX-2 gene expression.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HVSMC culture and incubations. HVSMC were grown by the explant technique from unused portions of saphenous veins harvested for coronary artery bypass surgery (New England Medical Center) as previously described (7). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), glutamine, penicillin, and streptomycin, passaged every 7-30 days by harvesting with trypsin-EDTA, and plated at a 1:3 ratio. For experiments, HVSMC (passages 2-6) were incubated with or without human recombinant IL-1beta for 6 h. In some experiments, inhibitors of arachidonate metabolism were added during the last 90 min of this incubation.

6-keto-PGF1alpha production. The stable metabolite of PGI2, 6-keto-PGF1alpha , was measured in VSMC supernatants by radioimmunoassay using a commercial kit (Advanced Magnetics, Cambridge, MA). The antibody used to detect 6-keto-PGF1alpha cross-reacts 7.8% with PGF1alpha , 6.8% with 6-keto-PGE1, 2.2% with PGF2alpha , 0.7% with PGE1, and 0.6% with PGE2.

Cellular levels of cAMP. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM) was added 20 min before cell extracts were obtained for cAMP determination. cAMP was extracted from the cells by rapid aspiration of the medium and addition of ice-cold 80% ethanol to each well. Cells were incubated at least 20 min on ice, and extracts were stored at -20°C. Extracts were centrifuged and the supernatants vacuum-dried and reconstituted in 50 mM sodium acetate (pH 6.2). The cAMP content of cell extracts was determined by radioimmunoassay using a cAMP antibody and 125I-labeled cAMP from Biomedical Technologies (Stoughton, MA). Cross-reactivity of cGMP in the cAMP assay was 0.017%.

RT-PCR. Total RNA was isolated using RNAzol B (Biotecx Laboratories, Houston, TX). RNA (2 µg) was reverse-transcribed for 1 h at 37°C with 200 U of MuMLV reverse transcriptase (GIBCO BRL), using an oligo(dT) primer and the buffer supplied by the manufacturer, in a total volume of 20 µl. The reaction was terminated by heating to 95°C for 5 min, and 2 µl of this first-strand cDNA was added to each PCR reaction. PCR mixes contained 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 1 mM MgCl2 (except cPLA2 PCR reactions, which included 1.5 mM MgCl2), 0.01 mg/ml gelatin, 200 µM of each dNTP, 1.5 µCi of [32P]dCTP, 250 nM of each primer, and 1 U Taq DNA polymerase (GIBCO BRL) in a total volume of 20 µl. Amplification was performed as previously described (7) with the PCR primers and annealing temperatures shown in Table 1. To enhance specificity, cDNA samples were heated to 95°C before the addition of Taq DNA polymerase for amplification employing sPLA2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. Each sample was assayed in triplicate, and each replicate was amplified for a different number of cycles within the exponential range of amplification. Products were size separated by electrophoresis in a 5% polyacrylamide gel and visualized by ethidium bromide staining. PCR bands were quantitated by cutting the relevant band from the gel and counting in a liquid scintillation counter. The relative amount of PCR product ([32P]dCTP incorporated) was normalized to the corresponding amount of GAPDH PCR product, and values were expressed relative to the control sample.

                              
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  		Table 1.   PCR primers

Western blot analysis. HVSMC in 100-mm dishes were incubated in DMEM supplemented with 0.5% FCS with or without IL-1beta (2 ng/ml) for 6 or 24 h. Cells were washed two times in ice-cold PBS, lysed in 85°C SDS-loading buffer, and boiled at 95°C for 5 min. Protein (50 µg/lane) was separated on a 6% SDS-polyacrylamide gel and transferred to nitrocellulose membranes using an electrotransfer apparatus (Trans blot cell; Bio-Rad). The membranes were blocked in Tris-buffered saline containing 5% nonfat dry milk and 0.05% Tween 20 and then incubated sequentially with anti-human COX-2 rabbit IgG (Cayman Chemical, Ann Arbor, MI), biotinylated goat anti-rabbit IgG, and streptavidin-biotinylated alkaline phosphatase complex (Bio-Rad). Blots were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

Drugs. Human recombinant IL-1beta was a gift from Dr. Richard Dondero (Cistron Biotechnology, Pine Brook, NJ). NS-398 was obtained from Cayman Chemical (Ann Arbor, MI). AACOCF3, oleyloxyethyl phosphorylcholine (OOPC), and thioetheramide phosphorylcholine (TEAPC) were obtained from Biomol (Plymouth Meeting, PA). Cycloheximide and actinomycin D were obtained from Sigma (St. Louis, MO). In some cases, drugs were dissolved in DMSO, and control cultures contained equivalent amounts of DMSO that never exceeded 0.1%.

Data analysis. Significant differences between groups were determined by unpaired t-test. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IL-1beta induces rapid increases in COX-2 mRNA and protein in HVSMC. mRNA encoding COX-1 was not detectable in either control or IL-1beta -stimulated HVSMC by a sensitive RT-PCR analysis, even after 30 cycles of amplification (data not shown). In contrast, COX-2 mRNA was detectable in HVSMC before stimulation and increased rapidly after stimulation with IL-1beta (2 ng/ml) (Fig. 1). Levels of COX-2 mRNA increased 16-fold after 1 h of incubation with IL-1beta and continued to increase to 50- to 60-fold at 8-24 h (Table 2), as determined by 32P quantitation of the relevant bands. (Although the 300-bp COX-2 PCR product is not visible in the 0-h sample in the photographic reproduction shown in Fig. 1, the band was clearly visible on the original gel and was also detectable by liquid scintillation counting.) HVSMC were incubated with IL-1beta in the presence of 5% FCS in this time-course study to reproduce the conditions used previously in our laboratory for studies of IL-1-induced prostanoid and cAMP synthesis (6). Incubation with 5% FCS alone did not induce COX-2 mRNA expression (data not shown). IL-1beta also induced COX-2 gene expression in HVSMC that were serum deprived before and during the incubation with IL-1beta , and the effect of IL-1beta was concentration dependent (Fig. 2). COX-2 mRNA remained detectable after 24 h of serum deprivation, and mRNA levels were not affected by 2 pg/ml IL-1beta , increased 4-fold in cells exposed to 20 pg/ml IL-1beta for 24 h, and increased 13-fold in cells exposed to 20 ng/ml IL-1beta (Fig. 2 and Table 2).


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  		Fig. 1.   Interleukin (IL)-1beta induces a rapid increase in cyclooxygenase (COX)-2 mRNA in human saphenous vein vascular smooth muscle cells (HVSMC). HVSMC were incubated with IL-1beta (2 ng/ml) in DMEM supplemented with 5% FCS for times shown, and levels of COX-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were assessed by RT-PCR (30 and 22 cycles, respectively). Molecular size markers (Hae III-digested ØX-174) are shown in left lane. COX-2 PCR products run near the 310-bp fragment.

                              
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  		Table 2.   Quantitation of COX-2 and cPLA2 mRNA levels in HVSMC



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  		Fig. 2.   Concentration dependence of IL-1beta -induced COX-2 and cPLA2 mRNA in HVSMC. HVSMC were serum deprived for 24 h in DMEM-F12 supplemented with insulin and transferrin (DMEM-F12-IT), and then RNA was prepared from 1 of the plates (lane 1). Remaining plates were incubated 24 h in fresh DMEM-F12-IT with or without IL-1beta (0, 0.002, 0.02, 0.2, 2.0, and 20 ng/ml; lanes 2-7, respectively). Levels of COX-2, cytosolic phospholipase A2 (cPLA2), and GAPDH mRNA were assessed by RT-PCR (30, 27, and 20 cycles, respectively). Similar results were obtained with HVSMC derived from a different patient.

Exposure to IL-1beta also induced expression of COX-2 protein, as determined by Western blot analysis (Fig. 3). COX-2 protein was not detectable in HVSMC incubated 6 or 24 h in medium alone, whereas a band with apparent molecular mass of ~68 kDa was present in cell extracts from HVSMC incubated 6 or 24 h with 2 ng/ml IL-1beta . Development of a replicate blot for a longer time in substrate revealed that HVSMC incubated with medium alone also expressed COX-2, but at levels that were lower than those in IL-1beta -treated HVSMC (data not shown). A band that migrated slightly faster than COX-2 was also apparent but was found to be nonspecific because it also appeared on an identical blot that was incubated with secondary antibody only, without prior incubation with COX-2-specific antibody (data not shown).


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  		Fig. 3.   IL-1beta induces expression of COX-2 protein in HVSMC. HVSMC were incubated in DMEM supplemented with 0.5% FCS with (+) or without (-) IL-1beta (2 ng/ml) for 6 or 24 h, and whole cell lysates were prepared. Equal amounts of cell protein (50 µg/lane) were separated on a 6% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were analyzed by Western blot analysis using human COX-2-specific antibodies. COX-2 band migrated with an approximate molecular mass of 68 kDa relative to biotinylated protein standards. Nonspecific (NS) band also appeared on an identical blot that was incubated with second antibody alone (not shown).

IL-1beta induces delayed increases in cPLA2 mRNA in HVSMC. Amplification of cDNA from HVSMC with primers specific for cPLA2 cDNA yielded PCR product of the expected size (490 bp) (Fig. 4). Levels of cPLA2 mRNA were low but detectable before stimulation with IL-1beta . Although the band obtained after 26 cycles of amplification was not visible by ethidium bromide staining (Fig. 4), it was detectable by liquid scintillation counting and also became visible after 28 cycles of amplification (data not shown). The levels of cPLA2 mRNA were unchanged 4 h after exposure to IL-1beta but were increased 5- to 6-fold after 8-24 h of exposure (Fig. 4 and Table 2). In a replicate experiment, cPLA2 mRNA increased 3.2-fold after a 24-h incubation with 5% FCS alone and 6.9-fold after a 24-h incubation with 5% FCS and IL-1beta (data not shown). IL-1beta also induced a delayed increase in cPLA2 mRNA in HVSMC that were serum deprived for 24 h before and during exposure to IL-1beta . HVSMC that had been serum deprived for 24 h also expressed cPLA2 mRNA before stimulation with IL-1beta (Fig. 2). cPLA2 mRNA levels were not affected by 2 pg/ml IL-1beta , increased 1.7-fold in HVSMC incubated 24 h with 20 pg/ml IL-1beta , and increased 2-fold in cells exposed to 20 ng/ml IL-1beta (Fig. 2 and Table 2). Western blot analysis verified that whole cell lysates from HVSMC contained cPLA2 that migrated with an apparent molecular mass of ~85 kDa. However, the level of cPLA2 was not detectably different in IL-1beta -treated versus nontreated HVSMC (data not shown).


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  		Fig. 4.   IL-1beta induces delayed increases in cPLA2 mRNA and does not affect secretory PLA2 (sPLA2) mRNA levels in HVSMC. HVSMC were incubated with IL-1beta (2 ng/ml) in DMEM containing 5% FCS for times shown. Levels of cPLA2, sPLA2, and GAPDH mRNA were assessed by RT-PCR (26, 28, and 22 cycles, respectively). cPLA2 and sPLA2 PCR products run between the 603- and 310-bp size markers shown in left lane.

sPLA2 mRNA and PGI2 synthase are constitutively expressed in HVSMC and not affected by IL-1beta . HVSMC contained abundant levels of mRNA for secreted PLA2. Substantial PCR product of the expected size (435 bp) was obtained from nontreated HVSMC after 28 cycles of PCR amplification (Fig. 4). Also, the levels of sPLA2 mRNA were not significantly affected by exposing HVSMC to IL-1beta for 1-24 h.

HVSMC also constitutively expressed abundant mRNA for PGI2 synthase. Substantial PCR product of the expected size (388 bp) was detectable in control samples after 29 cycles of amplification (Fig. 5). The levels of PGI2 synthase mRNA were not affected by exposure to IL-1beta , as determined by quantitation of the PCR product relative to GAPDH. Similar results were obtained with HVSMC derived from a different patient.


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  		Fig. 5.   IL-1beta does not affect PGI2 synthase mRNA levels in HVSMC. HVSMC were incubated with IL-1beta (2 ng/ml) in DMEM supplemented with 5% FCS for times shown, and levels of PGI2 synthase and GAPDH mRNA were assessed by RT-PCR (29 and 20 cycles, respectively). PGI2 synthase PCR product runs between the 603- and 310-bp size markers shown in left lane. Similar results were obtained with HVSMC derived from another patient.

IL-1-induced cAMP in HVSMC is blocked by protein synthesis inhibitors. Previous studies from our laboratory indicate that IL-1-induction of prostanoid synthesis in HVSMC results in activation of adenylate cyclase, and thus intracellular cAMP levels serve as a marker of IL-1-induced prostanoid synthesis (6). In the present study, HVSMC exposed to IL-1beta for 6 h had elevated intracellular levels of cAMP, measured in the presence of the phosphodiesterase inhibitor IBMX (Fig. 6), as previously reported (6). Furthermore, IL-1-induced cAMP production was blocked by an inhibitor of mRNA synthesis, actinomycin D (2 µg/ml), and by an inhibitor of protein translation, cycloheximide (20 µg/ml), supporting the hypothesis that the elevated prostanoid synthesis in HVSMC treated with IL-1beta for 6 h is dependent on the synthesis of new mRNA and protein.


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  		Fig. 6.   IL-1beta -induced cAMP in HVSMC is blocked by inhibitors of protein and mRNA synthesis. HVSMC were incubated in DMEM supplemented with 5% FCS without (Con) or with IL-1beta (2 ng/ml), cycloheximide (Cyclohex; 20 µg/ml), or actinomycin D (Act D; 2 µg/ml). IBMX (1 mM) was added to each well 30 min before cAMP determination. * P < 0.005, significantly different from control HVSMC.

In experiments that assessed the effect of protein, COX, or PLA2 inhibitors on IL-1-induced cAMP, HVSMC were incubated with IL-1beta in medium that contained 5% FCS, as in previous studies (6). FCS was included because low levels of DMSO used to solubilize some of the inhibitors were cytotoxic to some HVSMC cell lines incubated in low-serum medium. IL-1beta induced cAMP to a similar degree in HVSMC that were incubated 6 h with IL-1beta in medium containing either 0.5% FCS (control: 3.17 pmol/well; IL-1beta -treated: 27.8 pmol/well) or no FCS (control: 1.46 pmol/well; IL-1beta -treated: 25.8 pmol/well).

NS-398 reverses IL-1beta -induced PGI2 and cAMP production. NS-398, a potent and selective inhibitor of COX-2, reversed IL-1beta -induced PGI2 synthesis. NS-398 was added during the last 90 min of the 6-h incubation with IL-1beta to reverse, rather than prevent, IL-1beta -induced PGI2 and cAMP production. NS-398 (10 µM) inhibited the accumulation of 6-keto-PGF1alpha in the medium of control cells and nearly abolished the IL-1beta -induced accumulation of 6-keto-PGF1alpha (Fig. 7A). NS-398 (10 µM) also reduced cAMP production in control HVSMC and almost abolished IL-1beta -induced cAMP production (Fig. 7B). NS-398 at 1 µM was nearly as effective as NS-398 at 10 µM. Indomethacin (10 µM) had the same effect as NS-398 on control and IL-1beta -stimulated 6-keto-PGF1alpha and cAMP production (Fig. 7, A and B).


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  		Fig. 7.   NS-398 reverses IL-1beta -induced 6-keto-PGF1alpha and cAMP production. HVSMC were incubated in DMEM supplemented with 5% FCS without (Con) or with IL-1beta (2 ng/ml). NS-398 or indomethacin (Indo) was added after 4.5 h, and media were changed to DMEM containing IBMX (1 mM) with or without NS-398 or Indo after 5.5 h. At 6 h, supernatants were obtained for measurement of 6-keto-PGF1alpha (A) and cells were extracted for determination of cAMP (B). Similar results were obtained in 2 additional experiments with HVSMC derived from different patients. * P < 0.002, significantly different from control HVSMC.

NS-398 attenuates IL-1beta -induced cPLA2 mRNA. Because enhanced PG production preceded the induction of cPLA2 mRNA, the effect of preventing IL-1beta -induced PG production with NS-398 on the subsequent increase in cPLA2 mRNA was determined. cPLA2 mRNA was increased 3.5-fold in HVSMC incubated 24 h with IL-1beta (2 ng/ml) compared with HVSMC incubated without IL-1beta for 24 h (Fig. 8). In contrast, cPLA2 mRNA was increased only 1.7-fold in HVSMC incubated 24 h with IL-1beta in the presence of NS-398 (10 µM). NS-398 alone did not affect the levels of cPLA2 mRNA. IL-1beta induction of cPLA2 mRNA was likewise attenuated in a similar experiment in which prostanoid synthesis was inhibited with indomethacin (data not shown).


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  		Fig. 8.   NS-398 attenuates IL-1beta -induced cPLA2 mRNA. HVSMC were serum deprived in DMEM-F12-IT overnight and then incubated in fresh DMEM-F12-IT without (lanes 1-4) or with (lanes 5-8) NS-398 (10 µM) for 1 h. IL-1beta (2 ng/ml) was then added to some of the plates (lanes 3, 4, 7, and 8), and HVSMC were incubated an additional 24 h. Levels of cPLA2 and GAPDH mRNA were assessed by RT-PCR (26 and 20 cycles, respectively). Quantitation of cPLA2 PCR product (normalized to GAPDH and relative to control in lanes 1 and 2): lanes 3 and 4, 3.51 (IL-1beta ); lanes 5 and 6, 1.07 (NS-398); lanes 7 and 8, 1.83 (NS-398 and IL-1beta ). Similar results were obtained in a replicate experiment in which COX was inhibited with Indo.

IL-1-induced cAMP is reversed by inhibition of cPLA2. cAMP accumulation in HVSMC treated with IL-1beta for 6 h was reversed by a 3-h incubation with the selective PLA2 inhibitor AACOCF3 (10-30 µM), which inhibits cPLA2 as well as sPLA2 (33). AACOCF3 (30 µM) attenuated IL-1beta -induced cAMP by 91% in the experiment shown in Fig. 9A and by 66% in a replicate experiment with HVSMC derived from a different patient (data not shown). cAMP accumulation in HVSMC treated with IL-1beta for 6 h was also attenuated by a 90-min incubation with the nonselective PLA2 inhibitor OOPC (10 µM) (Fig. 9B), which inhibits cPLA2 as well as sPLA2 (33). In six experiments with HVSMC derived from different patients, OOPC attenuated IL-1beta -induced cAMP by 72 ± 7%. In contrast, a 90-min exposure to TEAPC (10 µM), which inhibits sPLA2 preferentially over cPLA2 (33), did not affect IL-1beta -induced cAMP (Fig. 9B). In four experiments with HVSMC derived from different patients, IL-1beta -induced cAMP production was unchanged by TEAPC (0 ± 12%).


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  		Fig. 9.   Inhibition of cPLA2 attenuates IL-1beta -induced cAMP production. HVSMC were incubated 6 h in DMEM supplemented with 5% FCS without (Con) or with IL-1beta (2 ng/ml). AACOCF3 or vehicle was added 3 h before extraction (A), or oleyloxyethyl phosphorylcholine (OOPC), thioetheramide phosphorylcholine (TEAPC), or vehicle was added 90 min before extraction (B) of cells for cAMP content. IBMX (1 mM) was added to each well 20 min before extraction. Results are representative of 2 experiments with AACOCF3, 6 experiments with OOPC, and 4 experiments with TEAPC. * P < 0.0005, significantly different from control HVSMC. + P < 0.005, significantly different from IL-1beta -treated HVSMC incubated without inhibitors.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IL-1 is a potent inducer of prostanoid production in VSMC derived from human blood vessels including the aorta, renal artery, and saphenous vein (1, 6, 27). The primary goal of the present study was to determine the mechanisms by which IL-1 upregulates prostanoid synthesis in HVSMC. A primary finding was that IL-1 induced a rapid and marked upregulation of COX-2 mRNA in HVSMC. Similar rapid induction of COX-2 has been observed in several cell types, including rabbit chondrocytes (30), rat mesangial cells (47), rat vascular smooth muscle cells (12), human monocytes (39), and human endothelial cells (22, 53). Induction of COX-2 mRNA was rapid, marked after only 1 h of stimulation, and preceded the induction of prostanoid synthesis in HVSMC that occurred 1.5-6 h after exposure to IL-1 (6). Levels of COX-2 protein were also increased in HVSMC exposed to IL-1 for 6 or 24 h. The present study focused on regulation of COX-2 mRNA expression; however, COX-2 may also be regulated at the translational level. mRNA encoding COX-1, the constitutive form of cyclooxygenase, was not present in control or IL-1-treated HVSMC, as indicated by highly sensitive RT-PCR analysis, in agreement with earlier findings (18). Furthermore, IL-1-induced PGI2 synthesis was reversed by the selective COX-2 inhibitor NS-398 at low concentrations (1 µM) that do not inhibit COX-1 (3). These results indicate that IL-1 induces prostanoid production in HVSMC via rapid induction of COX-2. A recent study (8) documents that IL-1 likewise upregulates COX-2 mRNA levels in freshly isolated segments of human saphenous vein and that IL-1-induced prostanoid production is blocked by NS-398.

Activation of adenylate cyclase is one of the downstream pathways activated by prostanoid receptor signaling. In previous studies, IL-1 markedly stimulated cAMP production in HVSMC and IL-1-induced cAMP synthesis was reversed by indomethacin (6), a cyclooxygenase inhibitor that inhibits both COX-1 and COX-2. In the present study, NS-398 reversed IL-1-induced cAMP. These results further document that IL-1 induces adenylate cyclase activity in HVSMC by inducing COX-2 gene expression, with subsequent generation of COX-2-derived prostanoids. Also, because adenylate cyclase is rapidly modulated by prostanoids, the level of IL-1-induced cAMP is an indicator of the rate of ongoing COX-2-derived prostanoid synthesis in HVSMC.

In previous studies, IL-1 markedly increased synthesis of PGI2 in HVSMC (6), and PGI2 was a potent inducer of cAMP production (6). cAMP synthesis was markedly stimulated by 1-10 nM PGI2 (levels similar to the levels of 6-keto-PGF1alpha produced by IL-1-treated cells). In comparison, PGE2 was a weak inducer of cAMP, having little effect on cAMP at 1-10 nM. In addition, IL-1-induced cAMP production was reversed by tranylcypromine, a PGI2 synthetase inhibitor. A previous report has suggested that IL-1 may also regulate PGI2 synthase gene expression and thereby specifically upregulate synthesis of PGI2. PGI2 synthase mRNA was increased twofold in human aortic endothelial cells exposed to IL-1 for 24 h (34). Therefore, we tested whether IL-1 induces PGI2 synthase mRNA in HVSMC. In the present study, PGI2 synthase mRNA was present constitutively in HVSMC and was not upregulated 1-24 h after stimulation with IL-1. Thus we obtained no evidence for specific upregulation of PGI2 synthesis at the level of PGI2 synthase gene expression.

In human fibroblasts, IL-1-induced prostanoid synthesis has been attributed to the upregulation of cPLA2 protein levels and the concomitant increase in activity, because prostanoid synthesis increased in the absence of induction of either COX-1 or COX-2 (29) and concurrent with induction of cPLA2 activity (20, 29). In both human fibroblasts and rat mesangial cells, IL-1 induction of cPLA2 mRNA and protein was delayed relative to the rapid induction of COX-2, occurring 8-24 h after stimulation with IL-1 (16, 20, 29). In the present study, IL-1 induced increases in the levels of mRNA encoding cPLA2 in HVSMC, and this increase was also delayed compared with the induction of COX-2 and prostanoid synthesis, occurring after 8 h of stimulation with IL-1. However, increases in cPLA2 protein levels were not detected. Thus increases in the level of cPLA2 protein do not account for the initial phase of IL-1-induced prostanoid synthesis in HVSMC, which begins as early as 1.5 h after stimulation with IL-1 and is marked after 6 h of stimulation (6). Although increases in cPLA2 protein were not detected in HVSMC in the present study, it is possible that delayed induction of cPLA2 occurs in vivo and contributes to the later stages of IL-1-induced prostanoid synthesis by providing additional arachidonate required for continued enhanced prostanoid synthesis.

Several recent studies have provided evidence of functional cross talk between PLA2 and COX enzymes. For example, cPLA2 is required for cytokine-induced expression of type II sPLA2 in a rat fibroblast cell line (26). Also, in mouse osteoblastic cells, prostanoids produced early after activation with IL-1 or tumor necrosis factor act in an autocrine fashion to enhance the delayed expression of both cPLA2 and COX-2 (35). Induction of cPLA2 gene expression in HVSMC occurred several hours after the induction of COX-2 gene expression. Therefore, we tested whether the delayed upregulation of cPLA2 mRNA was either mediated or enhanced by the initial COX-2-mediated upregulation of prostanoid synthesis. IL-1-induced cPLA2 was attenuated in HVSMC in which COX-2 activity was inhibited by treatment with NS-398. These results suggest that the initial phase of IL-1-induced prostanoid synthesis, which is mediated by upregulation of COX-2, amplifies the delayed upregulation of cPLA2 mRNA, which in turn may contribute to the later phase of prostanoid synthesis. The present study did not address how the initial upregulation of COX-2 enhances the delayed upregulation of cPLA2. It is possible that IL-1-induced cAMP upregulates cPLA2 gene expression. Alternatively, cAMP-independent effects of prostanoids may mediate the delayed upregulation of cPLA2. In this regard, IL-1-induced PGE2 was shown to amplify the delayed expression of cPLA2 in mouse osteoblastic cells (35). It is also possible that the effect is indirect. For example, cPLA2 expression may be somehow regulated by availability of arachidonic acid such that the initial increase in prostanoid synthesis depletes arachidonic acid, which in turn upregulates cPLA2 expression. Thus these studies further support the concept of functional cross talk between PLA2 and COX enzymes.

In several different cell types that have been studied, including rat aortic VSMC (37), rat mesangial cells (38), or rat astrocytes (40), sPLA2 mRNA is not expressed constitutively, but its expression is markedly increased after exposure to IL-1. In HVSMC, mRNA encoding sPLA2 was constitutively expressed, and its expression was not affected by IL-1. These results support the observation that the regulation of sPLA2 gene expression is highly cell-type specific (24). The differential expression of the sPLA2 gene in rat aortic VSMC versus human saphenous vein SMC may represent differences between VSMC derived from different blood vessels. Alternatively, sPLA2 gene expression may differ in human versus rat VSMC. In support of the latter hypothesis, SMC located within normal human uterine arteries and in atherosclerotic lesions of human abdominal aorta and carotid artery have been shown to contain immunoreactive sPLA2 (21). Thus constitutive production of sPLA2 may be a property of normal HVSMC in vitro as well as in vivo.

Several studies have focused on the respective roles of cPLA2 and sPLA2 in the regulation of prostanoid synthesis. sPLA2s are released from cells and are activated by the high levels of Ca2+ encountered, thereby releasing arachidonate and other lipids from the extracellular surface of the plasma membrane (41) of either the same cell or neighboring or distant cells. In contrast, cPLA2 generates arachidonic acid within cells (9, 10, 17, 25). The present study employed selective and nonselective inhibitors of PLA2 to establish their respective roles in IL-1-induced prostanoid synthesis in HVSMC. IL-1-induced cAMP was reversed by a 90-min incubation with AACOCF3, a trifluoromethyl ketone analog of arachidonic acid that is a slow-binding, yet specific and potent, inhibitor of cPLA2. AACOCF3 is four orders of magnitude more potent as an inhibitor of human cPLA2 than of nonpancreatic sPLA2 (52) and inhibits arachidonate release in human platelets (4, 46) and rat mesangial cells (16). IL-1-induced cAMP was also reversed by a nonselective inhibitor of PLA2, OOPC, which inhibits both secreted and cytosolic forms of PLA2 (31). In contrast, TEAPC, a preferential inhibitor of sPLA2 with little effect on cPLA2 at the concentrations used (31), did not attenuate IL-1-induced cAMP. Together, the data support the hypothesis that IL-1-induced prostanoid production requires arachidonate produced by the cytosolic rather than the secreted form of PLA2.

Many ligands, including platelet-derived growth factor, thrombin, and phorbol esters, elicit rapid activation of cPLA2 activity and prostanoid synthesis by increasing intracellular Ca2+, resulting in translocation of cPLA2 to the nuclear envelope and endoplasmic reticulum, where it is active (15, 48). These same ligands also induce phosphorylation of cPLA2 at Ser-505, leading to its activation (28, 46). The fact that cPLA2 inhibitors reversed IL-1-induced prostanoid synthesis in the present study indicates that cPLA2 was active in HVSMC that had been stimulated 6 h with IL-1. The present study did not address whether cPLA2 was constitutively active in HVSMC or whether IL-1 induced activation of cPLA2. It is possible that IL-1 induces rapid phosphorylation of cPLA2 in HVSMC, as previously shown in human fibroblasts (29) and rat mesangial cells (16). Alternatively, it is possible that IL-1 induces translocation of cPLA2 to the nuclear envelope and endoplasmic reticulum; however, IL-1-receptor activation has not been linked to increases in intracellular free Ca2+, which are thought to initiate translocation. Finally, it is possible that cPLA2 activity represents residual stimulation by low levels of serum mitogens, which may maintain sufficient levels of intracellular free Ca2+ and may also be sufficient to induce phosphorylation of cPLA2, as previously suggested (16, 20, 29).

It has recently been proposed that distinct COX isoforms are linked to distinct PLA2 isoforms and thus utilize distinct arachidonate pools. PG synthesis in activated fibroblasts and macrophages requires expression of COX-2; arachidonate released by these cells on activation cannot be utilized by COX-1, which is expressed constitutively by the cells (43). However, when fibroblasts are cocultured with activated mast cells that release sPLA2, arachidonic acid is released from the fibroblast membranes and is utilized by COX-1 present in fibroblasts for PG synthesis (44). In activated mast cells, two distinct COX-PLA2 pathways mediate temporally distinct phases of PG production. An early, transcellular pathway involves the release of sPLA2 by mast cells and subsequent mobilization of arachidonate extracellularly, which then becomes available to COX-1. A delayed, intracellular pathway involves cPLA2-catalyzed release of arachidonate intracellularly, which becomes available to COX-2, but not COX-1, for subsequent conversion to PG precursor within the cell (45). The results of the present study are consistent with a model of cPLA2 linkage to COX-2: IL-1 induces PG synthesis in HVSMC primarily via an intracellular pathway involving cPLA2 and COX-2. The present results further document that COX-2-generated prostanoid metabolites activate adenylate cyclase. Whether COX-2-generated prostanoids activate adenylate cyclase differentially from COX-1-generated prostanoids remains to be established.

Studies involving other cell types suggest that the specific linkage between COX and PLA2 isoforms is cell-type specific. Some cells demonstrate a functional linkage between sPLA2 and COX-2. sPLA2, anchored on cell surfaces via its heparin-binding domain, augments delayed COX-2-dependent PG synthesis in several different cell lines (26, 35, 36) and is crucial to COX-2-dependent PG synthesis in mouse mast cells (2). Conversely, immediate PG synthesis, which is elicited within minutes by ligands that mobilize intracellular Ca2+ and activate cPLA2, likely involves COX-1, which is expressed constitutively by many cells (46).

In the present study, COX-2, cPLA2, and protein synthesis inhibitors attenuated PGI2 and cAMP synthesis in HVSMC that were not stimulated with IL-1. Consistent with this finding, expression of COX-2 and cPLA2 mRNA were also detectable in the absence of IL-1 stimulation. However, the levels of COX-2 mRNA and prostanoid synthesis in nonstimulated cells were variable in different experiments, most likely due to differences in HVSMC cultures derived from different coronary bypass patients. It is possible that activation of the cPLA2-COX-2 pathway in the absence of IL-1 stimulation may represent activation by low levels of serum-derived mitogens. However, it is noteworthy that COX-2 and cPLA2 mRNA levels were not lower in HVSMC that had been serum deprived for 24 h, arguing against this possibility. Alternatively, basal expression of COX-2 and cPLA2 may represent serum-independent effects of culture in vitro. In support of the latter hypothesis, COX-2 was not observed in freshly isolated human saphenous vein segments but was expressed in this tissue after a 48-h incubation in serum-free medium in vitro (8). It was concluded that COX-2 expression resulted from surgical trauma to the vein; however, an alternative explanation is that COX-2 was induced by in vitro culture conditions other than serum.

Several lines of evidence support an important role of prostanoids in the systemic and local vasodilatory effects of IL-1. The present study delineates a pathway whereby IL-1 induces prostanoid production and thereby may inhibit contraction of HVSMC. IL-1 induces a rapid induction of COX-2 in HVSMC, and COX-2 utilizes a pool of arachidonate that is generated by the action of cPLA2. Furthermore, prostanoids, which are ultimately produced by the action of COX-2, are potent activators of adenylate cyclase in HVSMC. Selective inhibitors of COX-2 or cPLA2 may represent an effective clinical tool to reverse excessive vasodilatation that may occur in states of IL-1 excess.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-47569 and by a grant from Baxter Healthcare, Renal Division, Extramural Grant Program.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. Beasley, New England Medical Center, Box 172, 750 Washington St., Boston, MA 02111 (E-mail: debbie.beasley{at}es.nemc.org).

Received 9 April 1998; accepted in final form 9 December 1998.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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