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© 1999 American Heart Association, Inc.
Original Contribution |
Glucocorticoids Downregulate Cyclooxygenase-1 Gene Expression and Prostacyclin Synthesis in Fetal Pulmonary Artery Endothelium
From the Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, Tex.
Correspondence to Philip W. Shaul, MD, Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9063. E-mail pshaul{at}mednet.swmed.edu
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Abstract |
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Abstract—Prostacyclin (prostaglandin I2 [PGI2]) is a key mediator of pulmonary vascular function during early postnatal life, and its production in the pulmonary vasculature rises markedly during that period because of increasing expression of cyclooxygenase type 1 (COX-1). The postnatal rise in COX-1 may be due to the release of inhibition by glucocorticoids, since plasma glucocorticoid levels fall after birth and glucocorticoids decrease PGI2 synthesis in certain nonpulmonary cell types. We therefore studied the direct effects of dexamethasone (DEX) on COX-1 expression in early-passage ovine fetal pulmonary-artery endothelial cells (PAECs). DEX (10–10 to 10–6 mol/L) caused a dose-related decrease in COX-1 mRNA expression that was evident by 24 hours, was maximal at 10–6 mol/L (50% inhibition), and was not due to changes in mRNA stability. There was a parallel decline in COX-1 protein expression. COX-1 protein rose following DEX withdrawal, and DEX blunted the stimulatory effect of 17ß-estradiol on COX-1 expression. DEX alone (10–8 mol/L for 48 hours) caused a 93% fall in basal PGI2 production, and bradykinin- and A23187-stimulated PGI2 were diminished 96% and 94%, respectively. Similarly, PGI2 synthesis from arachidonic acid fell 86% with DEX; all of the above effects are consistent with COX-1 downregulation. The glucocorticoid receptor (GR) antagonist mifepristone (RU-486; 10–6 mol/L) blocked the inhibitory effect of DEX, and GR expression was evident by immunoblot analysis. These findings indicate that glucocorticoids downregulate COX-1 expression and PGI2 synthesis in fetal PAECs through the activation of PAEC GR and effects on COX-1 gene transcription. This mechanism may modulate pulmonary PGI2 production in the perinatal period, and it may also play a role in the effects of glucocorticoids on the systemic circulation at a variety of ages.
Key Words: cyclooxygenase-1 • endothelium • glucocorticoid • prostacyclin • pulmonary circulation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostacyclin (prostaglandin I2 [PGI2]) and other vasodilator prostaglandins are key mediators of pulmonary vascular function during early postnatal life. PGI2 administration in the newborn causes pulmonary vasodilation, and vasoconstriction occurs when its endogenous synthesis is inhibited. In addition, PGI2 attenuates hypoxic pulmonary vasoconstriction in the neonatal period.1 2 3 We have previously demonstrated that the rate-limiting enzyme in vascular PGI2 synthesis in the newborn lung is cyclooxygenase (COX), and that the capacity for vascular PGI2 production increases markedly during the early postnatal period. In addition, we have shown that this is due to an upregulation in the expression of the type 1 isoform of COX (COX-1).4
The mechanism underlying the developmental increase in COX-1 expression in the newborn pulmonary vasculature is unknown. However, the occurrence of the upregulation during a period of marked changes in hormonal and neuronal influences on cardiovascular homeostasis suggests that the upregulation is due to either increased effects of a factor that enhances COX-1 expression or decreased effects of a factor that attenuates COX-1 expression. One potential factor in the latter category is circulating glucocorticoids, which fall markedly in the newborn and which have been demonstrated to attenuate PGI2 synthesis in certain nonpulmonary cell types.5 6 7 8 9
Therefore, to delineate the basis for the maturational increase in COX-1 abundance in the newborn pulmonary circulation, the present studies were designed to determine the effects of glucocorticoids on COX-1 expression in fetal pulmonary-artery endothelial cells (PAECs). Experiments were performed in early-passage, cultured ovine fetal PAECs, which we have previously used in studies of estrogen-mediated effects on COX-1.10 The use of the PAECs allows for the evaluation of the direct effects of glucocorticoids on the pulmonary endothelium, avoiding the secondary effects that are possible in an intact animal model because of changes in pulmonary parenchymal and cardiac function.11 12 13 On the basis of the observation that pulmonary arterial COX-1 expression increases during the early postnatal period when newborn plasma glucocorticoid levels are falling5 6 and the evidence that glucocorticoids attenuate PGI2 synthesis in certain nonpulmonary cells,7 8 9 we hypothesized that glucocorticoids downregulate COX-1 expression in ovine fetal PAECs. In addition to testing this hypothesis, studies were performed to answer the following questions. (1) Is PGI2 synthesis attenuated in parallel with the effects on COX-1? (2) What is the time course of the effects of glucocorticoids? (3) What is the basis of the effects of glucocorticoids on COX-1 and PGI2 synthesis? (4) What are the combined effects of glucocorticoids and estrogen, which upregulates COX-1 expression in fetal PAECs10 ?
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Culture and Treatment
PAECs were obtained from mixed-breed fetal lambs at 125 to 135 days gestation (term=144±4 days) and propagated using methods we have previously reported.14 Near-confluent cells at passage 4 to 6 were placed in serum-free medium for 12 hours to remove the effects of serum-derived steroids. The cells were then placed in medium containing 20% charcoal-stripped serum. The charcoal stripping removes steroid hormones.15 The cells were treated for up to 48 hours with either control medium or medium containing various concentrations of dexamethasone (DEX) ranging from 10–10 to 10–6 mol/L. DEX treatment was repeated every 24 hours. Under pretreatment conditions, COX-1 expression is evident in the fetal PAECs and COX-2 is not detectable.16
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
Assay
A semiquantitative RT-PCR assay was used to evaluate COX-1 mRNA
abundance in the PAECs, because the mRNA was not detectable in the
PAECs by Northern analysis of poly(A)+
RNA. We have previously used this technique in studies of the effects
of estrogen on PAEC COX-1 mRNA levels.10 PCR was performed
using specific oligonucleotide primers for sheep
COX-1.10 17 The PCR products were size fractionated by
agarose gel electrophoresis, and their identity was confirmed and they
were quantified by Southern blotting and densitometric analysis
of the resulting autoradiographs. PCR product identity was also
confirmed by direct double-stranded sequencing. To control for the RT
step and for mRNA abundance and stability, RT-PCR was also done for the
housekeeping gene malate dehydrogenase (MDH) using published
oligonucleotide primer sequences.10 18 To
evaluate COX-1 mRNA stability, additional RT-PCR experiments were done
using cells treated with 25 µg/mL actinomycin D for varying time
periods up to 2 hours.
Immunoblot Analysis
To quantify the levels of COX-1 protein expression in control
and DEX-treated cells, immunoblot analysis was
performed using methods that generally followed those that we have
previously reported.10 14 The effects of DEX withdrawal
and the combined effects of DEX and 17ß-estradiol
(E2ß), which upregulates COX-1 expression in
PAECs,10 were also evaluated by immunoblot
analysis. Purified COX-1 protein (Cayman Chemical Co) was used
as a positive control. Similar techniques, including the use of a
positive control, were used to evaluate COX-2 protein expression. To
determine whether glucocorticoid receptor (GR) protein is expressed in
the ovine fetal PAECs, immunoblot analysis was also
performed using an antiserum (2 µg/mL) directed against the
DNA-binding domain of the mouse GR (clone BuGR 2, Affinity Bioreagents,
Inc, Golden, Colo). The secondary antibody was peroxidase-linked
anti-mouse antibody (Amersham).
Incubations for PGI2 Synthesis
PAECs grown in 24-well plates were incubated for determinations
of PGI2 synthesis over 60 minutes as previously
reported.10 To determine the reaction in the
PGI2 synthetic cascade that may be modified by
glucocorticoids, selected wells of PAECs were incubated in RPMI alone,
indicative of basal (nonstimulated) synthesis, and others were treated
with agents that activate the synthetic pathway at various
steps.10 Incubations were performed in the presence of
10–5 mol/L bradykinin to assess
PGI2 synthesis stimulated by receptor-mediated
mobilization of arachidonic acid from phospholipids by
phospholipase A2.19 Incubations with
the calcium ionophore A23187 (10–5 mol/L) were
performed to evaluate PGI2 production
stimulated by an increase in cytosolic free calcium, which
activates arachidonic acid mobilization through
phospholipase A2 by a non-receptor–mediated
process.20 Exogenous arachidonic
acid (10–5 mol/L) was used to stimulate
PGI2 synthesis to assess changes in COX
activity,21 bypassing potential changes in phospholipase
A2 expression or activity. Samples of cell
incubation media were assayed for the stable metabolite of
PGI2, 6-keto-prostaglandin
F1
(6-keto-PGF1) by
RIA as previously reported.14 In all experiments, n=4 to 6
for each determination, and findings were replicated in 2 or 3 studies
using cells from different primary cultures.
Statistical Analysis
ANOVA with Neuman-Keuls post hoc testing was used to compare
mean values between more than 2 groups. Nonparametric ANOVA
was used when indicated. Single comparisons between groups were
performed with nonpaired Student's t tests. Significance
was accepted at the 0.05 level of probability. All results are
expressed as mean±SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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COX-1 mRNA Expression
The effects of varying concentrations of DEX on steady-state levels of COX-1 mRNA in fetal PAECs are shown in Figure 1
. The cells were treated with control
medium or medium with 10–10 to
10–6 mol/L DEX for 48 hours. Single PCR
products were obtained for COX-1 at the expected size of 355 bp
(Figure 1A). The representative Southern blot
reveals a dose-dependent decrease in COX-1 mRNA abundance as determined
by RT-PCR in response to DEX treatment. PCR was also performed for MDH
to control for the RT step, yielding a single PCR product at the
expected size of 369 bp (Figure 1A). There was no change in MDH
mRNA abundance with DEX treatment. Quantitative densitometry for 3
independent experiments confirmed these results (Figure 1B).
There was a concentration-related decrease in steady-state COX-1 mRNA
levels, with a threshold concentration of 10–10
mol/L and a maximal effect of 50% diminution at
10–6 mol/L. Treatment with control medium alone
for 48 hours had no effect on either COX-1 or MDH mRNA abundance as
assessed by RT-PCR (data not shown).
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The time course of the effect of DEX on COX-1 mRNA expression in fetal
PAECs is given in Figure 2. In the
representative Southern blot (Figure 2A), COX-1
mRNA abundance as determined by RT-PCR was not altered by DEX
(10–8 mol/L) exposure for 12 hours, but the
effect was evident by 24 hours, and it persisted for at least 48 hours.
DEX had no effect on MDH mRNA abundance at any time point (Figure 2A). Quantitative densitometry for 3 independent experiments
confirmed these results, showing a 25% decrease in COX-1 expression at
24 hours and a persistent decrease of 38% at 48 hours.
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The effects of DEX on COX-1 mRNA stability are depicted in Figure 3. RT-PCR was performed on cells treated
with control medium or medium with 10–8 mol/L
DEX for 48 hours, followed by 25 µg/mL actinomycin D for varying
durations up to 2 hours. The representative study shown
reveals no difference in COX-1 mRNA degradation in control and
DEX-treated cells. In 4 independent experiments COX-1 mRNA half-life
was similar in control and DEX-treated cells, being 1.2±0.1 hours and
1.2±0.1 hours (mean±SEM), respectively.
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, dashed line) or medium
containing 10–8 mol/L DEX (
, solid line) for 48 hours,
followed by 25 µg/mL actinomycin D for varying time periods up to 2
hours. COX-1 mRNA abundance, assessed by RT-PCR, is expressed as
percentage at time 0 of actinomycin D. Linear regression
analysis yielded r=-0.98 for control
cells and r=-0.99 for DEX-treated cells.
Similar findings were obtained in 4 independent experiments.
COX Protein Expression
The effects of DEX on COX-1 protein expression are shown in Figure 4. In the representative
immunoblot provided (Figure 4A), COX-1 was decreased
in cells treated with 10–8 mol/L DEX for 48
hours. The cumulative findings of 3 independent experiments confirmed
these results (Figure 4B), revealing a 44% decrease in COX-1
protein with DEX treatment. COX-2 protein was not detected in either
control or DEX-treated cells (data not shown).
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PGI2 Synthesis
The findings for basal and stimulated PGI2
synthesis in control and DEX-treated cells are depicted in Figure 5. In numerous independent experiments,
basal PGI2 production in control cells
ranged from mean values of 349 to 1090 pg/well. In control cells,
bradykinin caused a 4-fold increase in PGI2
production, the calcium ionophore A23187 caused a 5-fold rise
in synthesis, and exogenous arachidonic acid caused a
13-fold increase in PGI2. Treatment with control
medium alone for 48 hours had no effect on either basal or stimulated
PGI2 synthesis (data not shown).
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The effects of DEX on basal PGI2 synthesis are
depicted in Figure 5A
. Following DEX treatment
(10–8 mol/L) for 48 hours, basal
PGI2 production was attenuated by 93%.
To determine whether this was due to effects on the production
of an endogenous agonist, the effects on synthesis
stimulated by bradykinin were evaluated (Figure 5B). Paralleling
the findings for basal synthesis, production with bradykinin
was attenuated 96%. To determine whether DEX causes alterations in
calcium-mediated mechanisms of arachidonic acid
release, PGI2 production with A23187 was
examined (Figure 5C). Similarly to the findings for
bradykinin-stimulated synthesis, PGI2
production with A23187 was diminished 94% by DEX. The effects
of DEX on arachidonic acid–stimulated
PGI2 synthesis are shown in Figure 5D.
Mimicking the results obtained with basal, bradykinin-stimulated, and
A23187-stimulated production, DEX caused an 86% decline in
PGI2 production with
arachidonic acid.
Role of GRs
To determine the role of GR in the effects of the hormone on
PGI2 synthesis in the PAECs, experiments were
performed in the presence or absence of the GR antagonist
mifepristone (RU-486). RU-486 had no effect on basal or stimulated
PGI2 synthesis in control cells (data not shown).
The diminution in basal PGI2 production
caused by DEX was fully reversed by GR inhibition (Figure 6A). Similarly, the DEX-mediated decline
in A23187-stimulated PGI2 synthesis was
completely negated by RU-486 (Figure 6B). Furthermore, the
effects of the steroid on arachidonic acid–stimulated
PGI2 production were also totally
reversed by the receptor antagonist (Figure 6C).
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To determine whether GRs are expressed in the fetal PAEC,
immunoblot analysis was performed. In the
representative immunoblot shown (Figure 7), signal for GR protein was detectable
at 98 kDa. Similar findings were obtained in 3 independent
experiments.
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DEX Withdrawal and Interaction With Estrogen
Additional studies were performed to evaluate the effects of DEX
withdrawal and the combined effects of DEX and
E2ß, which upregulates COX-1 expression in
PAECs10 (Figure 8). In the
representative immunoblot shown (Figure 8A), DEX exposure for 24 hours caused a decline in COX-1 protein
expression, and the ensuing withdrawal of DEX for 24 hours caused an
increase in COX-1. E2ß alone caused an increase
in COX-1 abundance, and combined exposure to DEX and
E2ß yielded COX-1 expression similar to levels
in control cells. The cumulative findings of 3 independent experiments
confirmed these results (Figure 8B). DEX alone for 24 hours
caused a 23% fall in COX-1, DEX withdrawal then led to a 2-fold
increase, E2ß alone yielded a doubling in
COX-1, and E2ß plus DEX resulted in COX-1
expression comparable to control values.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we have evaluated the effects of DEX on COX-1 expression in cultured ovine fetal PAECs. We have shown that DEX causes a decrease in COX-1 mRNA expression in these cells. To our knowledge, this is the first demonstration of a direct effect of glucocorticoids on COX-1 mRNA expression in endothelial cells.
We have also shown that the effect of DEX occurs at a threshold concentration of 10–10 mol/L and that there is maximal downregulation of >50% at 10–6 mol/L. In addition, the effect of glucocorticoids on COX-1 mRNA expression is not evident until 24 hours but persists for at least 48 hours. This time course contrasts with the findings of prior studies in nonendothelial cells, which have all entailed acute serum stimulation of COX-1 upregulation. In vascular smooth muscle cells, renal mesangial cells, and COX-1–transfected 3T3 cells, serum stimulation has been found to cause COX-1 upregulation within 3 hours, and this effect is blunted by glucocorticoid exposure.7 8 9 In contrast, in the present study the control, serum-containing medium had no effect on COX-1 mRNA, and the observed changes were in response to DEX alone. As such, the present findings are the first to demonstrate a direct effect of glucocorticoids on basal COX-1 mRNA expression in any cell type. Furthermore, COX-1 mRNA stability in the fetal PAEC is not altered by DEX exposure, suggesting that the effect is mediated at the level of COX-1 gene transcription.
The effects of DEX on COX-1 protein expression were also evaluated. Following exposure to 10–8 mol/L DEX for 48 hours, COX-1 protein was decreased by 44%, paralleling the decline in COX-1 mRNA. In contrast to COX-1, COX-2 protein was not detected in control cells, and it was not possible to evaluate the regulation of COX-2 under the conditions studied. Previous investigators have demonstrated that the induction of COX-2 in a variety of paradigms is attenuated by glucocorticoids.21
Studies were also performed to assess the effect of glucocorticoids on endothelial cell PGI2 synthesis. Following exposure to 10–8 mol/L DEX for 48 hours, basal PGI2 synthesis was attenuated by 93%, and bradykinin-stimulated production was decreased by 96%. These results indicate that the effect of glucocorticoids on PGI2 synthesis is not due to changes in the production of a local receptor agonist or nonspecific effects related to insertion of the steroid, which is similar in structure to cholesterol, into the cell membrane.21 In addition, PAEC PGI2 synthesis stimulated with A23187 was decreased by 94% following DEX treatment. This suggests that the effect of the hormone does not involve changes in agonist receptor density or function and that glucocorticoid-mediated changes in phospholipase A2 activity or expression must be considered.21 However, since PGI2 synthesis in the presence of excess arachidonic acid was also diminished following DEX treatment, and to a similar degree (decreased 86%), the effect of glucocorticoids is not mediated by changes in phospholipase A2 function. As such, these observations indicate a glucocorticoid-mediated effect on COX enzymatic activity, and this is consistent with the finding that glucocorticoids attenuate COX-1 mRNA and protein expression. Interestingly, the effects of DEX on PGI2 production (86% to 96% inhibition) exceed the degree of change in COX-1 expression (44% inhibition), suggesting that posttranslational mechanisms may also be involved. Taken collectively, these results are the first to demonstrate that glucocorticoids downregulate endothelial-cell COX-1 expression and activity. These findings contrast with those of previous studies in rabbit coronary microvascular endothelium and human umbilical vein endothelium, in which DEX inhibited basal and A23187-stimulated but not arachidonic acid-stimulated PGI2 synthesis; such results are consistent with effects proximal to COX.22 23 Thus, the present findings suggest unique effects of DEX on COX-1 in PAECs.
We also evaluated the role of endothelial GR in this process. GR antagonism with RU-486 completely reversed the attenuation in basal, A23187-stimulated, and arachidonic acid-stimulated PGI2 synthesis. This indicates that GR activation is necessary for the effects of the hormone on pulmonary endothelial COX-1 expression and function. Furthermore, immunoblot analysis revealed that GR protein is expressed in the fetal PAEC. Studies of adult human lung using in situ hybridization and immunohistochemistry have localized GR mRNA and protein to the pulmonary endothelium,24 but the present observations are the first to reveal that pulmonary endothelial GRs are functional.
A degree of caution may be appropriate in the direct extrapolation of the present findings in the cultured cells to processes in the intact lung. However, the use of the cultured cells allows us to evaluate the direct effects of single factors such as glucocorticoids on PAEC phenotype, thereby avoiding the secondary changes possible in an intact animal model resulting from known pulmonary parenchymal and cardiac effects of the hormone.11 12 13 In addition, we have previously demonstrated that numerous phenotypic characteristics regarding the regulation of COX-1 expression are conserved in cultured PAECs.14 16 Moreover, although the effect of cell culture on endothelial GR expression is not known, studies of estrogen receptors in human umbilical vein and bovine aortic endothelium suggest that steroid receptor expression is lost with the passage of a variety of endothelial cell types.25 As such, the use of the ovine fetal PAECs may be highly advantageous because GR expression is conserved. Thus, the observed effect of glucocorticoids on COX-1 expression and PGI2 synthesis in the PAEC may indeed reflect events occurring in the intact lung.
Keeping these potential limitations in mind, there are important physiological and pathophysiological implications of glucocorticoid-mediated regulation of pulmonary endothelial COX-1 expression. These are perhaps best represented by the findings obtained in the studies of the effects of E2ß with or without added DEX and DEX withdrawal. In the fetus, pulmonary vascular COX-1 expression rises at a time when plasma E2ß levels are increasing, paralleling the observed augmentation in PAEC COX-1 with exposure to E2ß alone.10 However, in the perinatal period there may also be interactions between COX-1 upregulation by E2ß and downregulation by DEX, as observed in the cultured PAECs exposed to both agents. Although not tested in the present study, the net effect of E2ß and glucocorticoids is most likely influenced by the relative concentrations of the hormones. Then, the further increase in pulmonary vascular COX-1 expression during the early newborn period, at a time when circulating glucocorticoid levels are falling,4 5 6 may be related to the removal of the inhibitory influence of glucocorticoids as seen in the PAECs following DEX withdrawal. Furthermore, the present findings suggest that in stress states in which glucocorticoid levels are abnormally elevated in the newborn,26 there may be a downregulation in pulmonary vascular COX-1 expression that may contribute to the pulmonary hypertension that is often associated with such conditions.
Along with the potential implications related specifically to the pulmonary circulation, the present observations are also highly relevant to the effects of glucocorticoids on systemic endothelial cell function. In terms of the perinatal period, Ibe et al27 have demonstrated that prenatal DEX therapy in sheep leads to decreased plasma PGI2 levels and attenuated PGI2 synthesis by isolated systemic arteries from the newborn lambs. The present observations suggest that this may be at least partly due to attenuated COX-1 expression in certain systemic vascular beds. This mechanism may explain the observation that systemic blood pressure is elevated in newborns following prenatal steroid therapy.28 In addition, the same mechanism may contribute to systemic hypertension in older patients receiving glucocorticoid therapy or those with Cushing syndrome.29 Further investigation of glucocorticoid-mediated regulation of COX-1 gene expression in the fetal PAEC will continue to advance our knowledge of the role of this hormone in both the pulmonary and systemic circulation.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL53546 and HD30276. We thank Marilyn Dixon for her assistance in preparing the manuscript.
Received July 2, 1998; accepted November 11, 1998.
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