Table Of ContentLinetal.CellCommunicationandSignaling2013,11:8
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RESEARCH Open Access
Up-regulation of COX-2/PGE by endothelin-1 via
2
κ
MAPK-dependent NF- B pathway in mouse brain
microvascular endothelial cells
Chih-Chung Lin1†, Hsi-Lung Hsieh2†, Ruey-Horng Shih3, Pei-Ling Chi3, Shin-Ei Cheng3 and Chuen-Mao Yang3*
Abstract
Background: Endothelin-1 (ET-1) is a proinflammatory mediator and elevated intheregions of several brain injury
and inflammatorydiseases.The deleterious effects ofET-1 on endothelial cells may aggravate brain inflammation
mediated through theregulationof cyclooxygenase-2 (COX-2)/prostaglandinE (PGE ) system invarious cell types.
2 2
However, thesignaling mechanismsunderlying ET-1-induced COX-2expression inbrainmicrovascular endothelial
cellsremain unclear. Herein we investigated theeffects of ET-1 inCOX-2 regulation in mouse brain microvascular
endothelial (bEnd.3) cells.
Results: The data obtained withWestern blotting, RT-PCR,and immunofluorescent staining analysesshowed that
ET-1-induced COX-2 expression was mediatedthrough an ET -dependent transcriptional activation. Engagementof
B
G-and G -protein-coupled ET receptors by ET-1 led to phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 and
i q B
then activated transcription factor NF-κB. Moreover, the data of chromatin immunoprecipitation (ChIP) and
promoter reporter assay demonstrated that the activated NF-κB was translocated into nucleus and bound to its
corresponding binding sites inCOX-2promoter,thereby turning on COX-2 gene transcription.Finally,up-regulation
ofCOX-2 by ET-1 promoted PGE release inthese cells.
2
Conclusions: These results suggestedthat inmouse bEnd.3 cells, activationof NF-κB by ET -dependent MAPK
B
cascades is essential for ET-1-induced up-regulationof COX-2/PGE system. Understandingthemechanismsof
2
COX-2 expression and PGE release regulated by ET-1/ET system onbrain microvascular endothelial cellsmay
2 B
provide rationally therapeutic interventions for brain injury or inflammatory diseases.
Keywords: Endothelin-1, COX-2,MAPK, NF-κB, Brain microvascular endothelial cells
Background endothelial cells are regulated at transcription and trans-
Cerebral capillary and microvascular endothelial cells lation levels by avariety of chemical and physical stimuli
play an active role in maintaining cerebral blood flow, and the levels of ET, ET-1 especially, are elevated in
microvascular tone and blood-brain barrier (BBB) func- shock, myocardial infarction, and kidney failure indica-
tions [1]. In the development of various vascular dis- tive of enhanced formation in these diseases [4]. More-
eases, an early finding is dysfunction of the vascular over, the bioactivity of ET-1 triggers vasoconstriction
endothelium that is closely related to clinical events in and pro-inflammatory action which have been impli-
patients with atherosclerosis and hypertension [2,3]. The cated in the pathogenesis of hypertension and vascular
vasoactive mediators such as endothelin (ET) could be diseases [3-6]. The effects of ET-1 are mediated through
produced by endothelial cells to maintain hemodynamic aGprotein-dependentregulation, includingtwo types of
responses. Production and release of ETs from cultured ET receptors: ET type A (ET ) and type B (ET ) [7].
A B
ET is involved in constriction and proliferation of
A
*Correspondence:[email protected] vascular smooth muscle cells, whereas ETB on endothe-
†Equalcontributors lial cells mediates the generation of nitric oxide, which
3DepartmentofPharmacology,CollegeofMedicine,ChangGungUniversity,
acts as vasodilator and inhibits platelet aggregation [8].
259Wen-Hwa1stRoadKwei-San,Tao-Yuan,Taiwan
Fulllistofauthorinformationisavailableattheendofthearticle Moreover, ET-1 also plays a substantial role in the
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normal development or in the central nervous system inflammatory mediators in neurodegenerative disor-
(CNS) diseases. In brain, endothelial cells [9] and astro- ders[19].
cytes [10] are potential sources of ET-1 release in re- ET-1 is known to activate ET receptors (ET or ET ),
A B
sponse to hypoxic/ischemic injury of the brain. A report a heterotrimeric G protein-coupled receptor (GPCR),
has shown that the ET receptors are located on brain which stimulate multiple signaling pathways and regu-
B
endothelial and vascular smooth muscle cells, and late diverse cellular functions [7,20-22]. The principal
modulate post-injury responses of these cells in the CNS mechanism underlying activation by ET-1 is mediated
[6].Thus,thereisanincreasinginterestintheregulatory through ET receptors coupling G proteins, resulting in
B q
roleofendothelialcellsinneurovascularcoupling,which activation of phospholipase C (PLC)-β, phosphoinositide
matches adequate supply of cerebral blood flow with the (PI) hydrolysis, and formation of inositol trisphosphate
local metabolic demands that are imposed by neural ac- (IP ) and diacylglycerol, leading to Ca2+ increase and
3
tivity [11]. As a fundamental component of the neuro- protein kinase C (PKC) activation [22]. Activation of a
vascular unit, endothelial dysfunction has been shown to G protein-coupled ET receptor has been also shown to
i B
be implicated in neurodegenerative diseases [11,12]. Cir- inhibit adenylylcyclase activity [23].Additionally, several
cumstantial evidence has further demonstrated that studies have demonstrated that activation of G and G
q i
overexpression of ET-1 on endothelial cells has deleteri- protein-coupled receptors via different signal pathways
ous effects on ischemic brain [1]. It has been demon- could activate diverse mitogen-activated protein kinases
strated that endothelial ET-1 induces cytokines or (MAPKs) [24]. It has been shown that ET-1-stimulated
chemokines (e.g., interleukine-1 or interleukin-8) pro- MAPKs activation to regulate various cellular responses
duction and secretion by non-neuronal cells, including including cell survival, growth, proliferation, and cellular
astrocytes and human brain-derived endothelial cells, hypertrophy in several cell types [21,25]. Several studies
which directly contributes to BBB breakdown during have suggested that up-regulation of COX-2 requires ac-
CNS inflammation [11,13]. These findings suggest that tivation of MAPKs and related transcription factors in
ET-1 might be involved in neuroinflammation. However, various cell types [22,26,27]. Our previous reports also
the detailed mechanisms responsible for ET-1 action are demonstrate that several GPCR agonists (e.g. sphingo-
still limited. sine 1-phosphate, thrombin, and bradykinin) stimulate
Cyclooxygenase (COX), known as prostaglandin- MAPKs and NF-κB activation associated with COX-2
endoperoxide synthase, is a rate-limiting key enzyme in expression in rat VSMCs and astrocytes [28,29]. Al-
the synthesis of prostaglandins (PGs). In this process, though several pro-inflammatory mediators have been
phospholipase A catalyzes the release of arachidonic extensively confirmed to rapidly up-regulate NF-κB-
2
acid (AA) from membrane phospholipids, while COX dependent genes such as COX-2 and play a critical role
catalyzes the conversion of AA into PGs [14,15]. COX in inflammation [29,30], the signaling mechanisms by
exists two isoforms: COX-1, which is constitutively which ET-1-induced MAPKs activation linked to COX-2
expressed under normal conditions in most tissues, expression and PGE production are not completely
2
mediates regulating normal physiological responses and defined inbrainmicrovascularendothelial cells.
controls vascular homeostasis; COX-2, is not detectable In this study, we investigated the molecular mechan-
in most normal tissues or cells, but its expression can be isms underlying ET-1-induced COX-2 expression in
induced by a variety of stimuli such as cytokines, endo- mouse brain microvascular endothelial (bEnd.3) cells.
toxin, and growth factors to produce PGs during inflam- These findings suggested that ET-1 induces COX-2 ex-
matory responses in various cell types like vascular pression at the transcriptional and translational levels,
endothelial and smooth muscle cells [16,17]. Previous which is mediated through the ET receptor (coupling
B
reports have shown that COX-2 immunoreactivity is a to G and G )-dependent activation of ERK1/2, p38
i q
characteristic finding in the synovial macrophage and MAPK, JNK1/2, and NF-κB pathway, leading to PGE
2
vascular cells of patients with arthritis and atheroscler- biosynthesis in mouse bEnd.3 cells. These results pro-
osis, respectively. Moreover, several studies have indi- vide new insights into the mechanisms of ET-1 action
cated COX-2 as a major therapeutic target for the which may be therapeutic value in brain inflammatory
treatment of inflammatory disorders like arthritis [14]. diseases.
The mice with homozygous deletion of the cox-2 gene
lead to a striking reduction of endotoxin-induced in- Results
flammation [18]. Accordingly, COX-2 may play a cru- ET-1inducesCOX-2expressionandPGE releasein
2
cial role in the development of various inflammatory bEnd.3cells
responses including vascular inflammation. In the To investigate the effect of ET-1 on COX-2/PGE sys-
2
CNS, several studies have indicated that up-regulation tem, bEnd.3 cells were incubated with various concen-
of COX-2 leads to production of PGs which are potent trations of ET-1 for the indicated time intervals. The
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data showed that ET-1 induced COX-2 expression in a biosynthesis, we collected the conditioned media and
time- and concentration-dependent manner (Figure 1A). determined PGE levels by using an EIA kit. The results
2
There was a significant increase within 2-4 h, reached a showed that ET-1 time-dependently stimulated PGE re-
2
maximal response within 6 h, and declined within 24 h. lease (Figure 1D) and a significant PGE production was
2
ET-1 also time-dependently induced COX-2 mRNA ex- observed within 4 h, reached a maximal response within
pression in bEnd.3 cells, determined by RT-PCR 6 h and slightly declined within 24 h. These results sug-
(Figure 1B). There was a significant increase in COX-2 gested that ET-1 induces COX-2/PGE system via up-
2
mRNA within 30 min, and reached a maximal response regulatingCOX-2gene expression inbEnd.3 cells.
within 2 h. Moreover, to confirm whether ET-1 induces
COX-2 expression via the transcription activity of COX- ET-1upregulatesCOX-2expressionviaanET receptor
B
2 promoter, cells were transiently transfected with COX- ET-1exertsits biological effects via ETreceptors,including
2 promoter-luciferase reporter construct and then sti- ET andET ,whicharemembersofGPCRsuperfamily[7].
A B
mulated with ET-1 for the indicated time intervals. As First, we determined which subtypes of ET receptors are
shown in Figure 1C, ET-1 time-dependently induced expressedonbEnd.3cellsbyRT-PCR.Thedatashowedthat
COX-2 promoter-luciferase activity in bEnd.3 cells. A ET but not ET receptors are expressed on bEnd.3 cells
B A
maximalresponsewasobtainedwithin4h.Ourprevious (Figure 2A). Next, to identify the subtypes of ET receptors
studies have shown that COX-2 expression induced by involved in ET-1-induced COX-2 expression, pretreatment
BK or sphingosine 1-phosphate is mainly responsible for with BQ-788 (an ET antagonist), but not BQ-123 (an ET
B A
prostanoid (PGE ) release in various cell types [28,29]. antagonist), attenuated the ET-1-induced COX-2 protein
2
Thus, to determine whether ET-1 could induce PGE (Figure 2B) and mRNA (Figure 2C) expression, suggesting
2
Figure1ET-1inducedCOX-2expressionandPGE release.(A)TimeandconcentrationdependenceofET-1-inducedCOX-2expression,cells
2
weretreatedwithvariousconcentrationET-1fortheindicatedtimeintervals.(B)TimedependenceofET-1-inducedCOX-2mRNAexpression,
cellsweretreatedwith10nMET-1fortheindicatedtimeintervals.COX-2mRNAwasanalyzedbyRT-PCR.(C)TimedependenceofET-1-induced
COX-2promotertranscriptionactivity,cellsweretransfectedaCOX-2promoter–luciferasereportergeneandthenincubatedwithET-1forthe
indicatedtimes.Thepromoterreporterassaywasperformedasdescribedin“MaterialsandMethods”.(D)PGE releaseinducedbyET-1,the
2
conditionedmediawerecollectedtoassayPGE levelbyEIAasdescribedin“MaterialsandMethods”(n=3ineachgroup;*P<0.05,#P<0.01
2
comparedwithvehicle).
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Figure2InvolvementofET receptorsinET-1-inducedCOX-2expression.(A)RT-PCRanalysisofETreceptorexpressioninbEnd.3cells.Lane
B
1:maker.Lane2:ET primersandbEnd.3RNA.Lane3:ET primersandbEnd.3RNA.(B,C)CellswerepretreatedwithBQ-123orBQ-788for1h
A B
andthenincubatedwithET-1for(B)6hand(C)1h.(D)CellsweretransfectedwithsiRNAforET receptorfor24handthenexposedtoET-1
B
for6h.The(B,D)COX-2proteinand(C)mRNAwereanalyzedbyWesternblotandRT-PCR,respectivelyasdescribedinFigure1.Dataare
expressedasmean±SEMofthreeindividualexperiments(n=3ineachgroup,#P<0.01ascomparedwithcellsstimulatedbyET-1alone).
that ET receptor is predominantly involved in these [7,22]. To further determine which of G proteins was
B
responses.Tofurtherconfirmthisnote,transfectionofcells involved in ET-1-induced COX-2 expression, pretreatment
with ET siRNA significantly down-regulated ET protein with either G protein antagonist GP antagonist-2 (GPA2)
B B i
expression and inhibited ET-1-induecd COX-2 expression or G protein antagonist GP antagonist-2A (GPA2A) con-
q
(Figure2D).ThesedatasuggestedthatET-1-inducedCOX- centration-dependently attenuated ET-1-induced COX-2
2 expression is mediated through an ET receptor- protein(Figure3A)andmRNA(Figure3B)expression.Fur-
B
dependentmannerinthesecells. thermore, to confirm these results, as shown in Figure 3C
and D,transfectionwith eitherGαorG αdown-regulated
i q
InvolvementofaG andG protein-coupledET receptor Gα or G α protein, respectively, and attenuated ET-1-
i q B i q
inET-1-induecdCOX-2expression induced COX-2 expression. These data demonstrated that
ET receptor has been shown to be a pleiotropic GPCR for ET-1-inducedCOX-2expressionismediatedthrougheither
ET-1 which is coupled to G proteins including G and G G orG protein-coupledET receptorsinbEnd.3cells.
i q i q B
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Figure3ET-1inducesCOX-2expressionviaaG andG proteins-coupledET receptor.(A,B)CellswerepretreatedwithG antagonist
i q B i
(GPA2)orG antagonist(GPA2A)for1handthenexposedtoET-1for(A)6hand(B)1h.(C,D)CellsweretransfectedwithsiRNAfor(C)Gαor
q i
(D)G αproteinfor24handthenexposedtoET-1for6h.The(A,C,D)COX-2proteinand(B)mRNAwereanalyzedbyWesternblotandRT-
q
PCR,respectivelyasdescribedinFigure1.Dataareexpressedasmean±SEMofthreeindividualexperiments(n=3ineachgroup,#P<0.01as
comparedwithET-1alone).
ET-1-inducedCOX-2expressionismediatedthrough 2phosphorylationwhichwasattenuatedbypretreatment
MAPKs with U0126, SB202190, or SP600125 during the period
Activation of MAPKs by ET-1 could modulate cellular of observation. Moreover, to ensure the roles of MAPKs
functions of endothelial cells [31]. To investigate the in ET-1-induced COX-2 expression, transfection with
roles of ERK1/2, p38 MAPK, and JNK1/2 in ET-1- siRNA of ERK2, p38 MAPK, or JNK1 down-regulated
induced COX-2 expression, pretreatment with the in- the expression of total ERK2, p38 MAPK, or JNK1 pro-
hibitor of MEK1/2 (U0126), p38 MAPK (SB202190), or tein and attenuated ET-1-induced COX-2 expression
JNK1/2 (SP600125) attenuated ET-1-induced COX-2 (Figure 4D). These data indicated that phosphorylation
protein (Figure 4A) and mRNA (Figure 4B) expression of ERK1/2, p38 MAPK, and JNK1/2 is involved in ET-1-
in bEnd.3 cells, suggesting the involvement of ERK1/2, induced COX-2 expression in bEnd.3 cells. To demon-
p38 MAPK, and JNK1/2 in ET-1-induced responses. To strate whether ET-1 stimulates ERK1/2, p38 MAPK, and
further determine whether ET-1-stimulated ERK1/2, p38 JNK1/2phosphorylation viaaGprotein-coupledET re-
B
MAPK, and JNK1/2 phosphorylation is involved in ceptor cascade, pretreatment with BQ-788, GPA2, or
COX-2 expression, as shown in Figure 4C, ET-1 time- GPA2A attenuated ET-1-stimulated ERK1/2, p38
dependently stimulated ERK1/2, p38 MAPK, and JNK1/ MAPK, and JNK1/2 phosphorylation during the period
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Figure4(Seelegendonnextpage.)
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(Seefigureonpreviouspage.)
Figure4ET-1-inducedCOX-2expressionismediatedthroughMAPKsphosphorylation.(A,B)Cellsweretreatedwith10nMET-1for(A)6
hand(B)1hintheabsenceorpresenceofU0126,SB202190,orSP600125.TheCOX-2proteinandmRNAexpressionweredeterminedby
WesternblotandRT-PCR.(C)TimedependenceofET-1-stimulatedERK1/2,p38MAPK,andJNK1/2phosphorylation,cellswereincubatedwith10
nMET-1fortheindicatedtimesintheabsenceorpresenceofU0126(1μM),SB202190(300nM),orSP600125(300nM).(D)Cellswere
transfectedwithsiRNAofERK2,p38MAPK,orJNK1andthenexposedtoET-1for6h.(E)CellswerepretreatedwithBQ-788(1μM),GPA2(1μM),
orGPA2A(1μM)for1handthenincubatedwithET-1(10nM)fortheindicatedtimes.ThecelllysateswerecollectedandanalyzedbyWestern
blottingusingananti-COX-2,anti-phospho-ERK1/2,anti-phospho-p38MAPK,anti-phospho-JNK1/2,anti-ERK2,anti-p38MAPK,anti-JNK1,oranti-
GAPDH(asaninternalcontrol)antibody.Dataareexpressedasmean±SEMofatleastthreeindividualexperiments(n=3ineachgroup;*P<0.05,
#P<0.01ascomparedwithET-1alone).
of observation (Figure 4E). These results demonstrated essential for ET-1-induced COX-2 gene up-regulation.
that G protein-coupled ET -dependent activation of The transcriptional activity of NF-κB was evaluated by a
(i/q) B
ERK1/2, p38 MAPK, and JNK1/2 by ET-1 is, at least in promoter (containing NF-κB binding sites)-luciferase ac-
part,requiredforCOX-2expression inbEnd.3cells. tivity assay. As shown in Figure 6A, ET-1 enhanced NF-
κB transcriptional activity in a time-dependent manner
NF-κBisrequiredforET-1-inducedCOX-2expression with a maximal response within 60 min, which was sig-
ET-1 has been shown to modulate cellular functions nificantly inhibited by pretreatment with an inhibitor of
through activation of NF-κB signaling in various cell NF-κB (Bay11-7082, 10 nM). Moreover, pretreatment
types [32]. To examine whether activation of NF-κB is with BQ-788 (1 μM), GPA2 (1 μM), GPA2A (1 μM),
required for ET-1-induced COX-2 expression, as shown U0126 (U0, 1 μM), SB202190 (SB, 300 nM), or
in Figure 5A and B, pretreatment with a selective NF-κB SP600125(SP,300nM)attenuatedNF-κBtranscriptional
inhibitor Bay11-7082, which blocks activation of NF-κB activity stimulated by ET-1 (Figure 6B), demonstrating
signaling, attenuated ET-1-induced COX-2 protein and that ET-1 enhances the NF-κB transcriptional activity
mRNA expression in bEnd.3 cells. To determine through an ET -dependent activation of MAPKs. Subse-
B
whether the involvement of NF-κB in ET-1-induced quently, we determined that ET-1-stimulates NF-κB p65
responses mediated through NF-κB (p65 subunit) trans- binding activity in a time-dependent manner by ChIP-
location, as shown in Figure 5C, ET-1 time-dependently PCR analysis (Figure 6C). ET-1-stimulated NF-κB p65
stimulated translocation of NF-κB p65 from cytosol into binding activity was inhibited by pretreatment with
nucleus determined by Western blot. A maximal re- U0126, SB202190, SP600125, Bay11-7082, or BQ-788
sponse was obtained within 90 min and sustained over (Figure 6C, lower part). In addition, we have demon-
120 min (upper part). Moreover, we also confirmed the strated that ET-1 time-dependently induces COX-2 pro-
NF-κB p65 translocation by an immunofluorescence moter activity (Figure 1C). We further demonstrated
staining. The imaging data confirmed that ET-1 stimu- that ET-1-increased the COX-2 promoter activity was
lated the p65 translocation at 90 min, which was inhib- significantly inhibited by pretreatment with BQ-788,
ited by pretreatment with Bay11-7082 (Figure 5C, lower GPA2, GPA2A, U0126, SB202190, SP600125, or Bay11-
part). We further demonstrated that ET-1 stimulated 7082 (Figure 6D), suggesting that ET-1 stimulates COX-
translocation of NF-κB p65 was attenuated by pretreat- 2 promoter activity via the ET -dependent activation of
B
ment with the inhibitor of ET receptor (BQ-788), MAPKs and NF-κB in bEnd.3 cells. To further ensure
B
MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 that NF-κB indeed mediates ET-1-induced COX-2 pro-
(SP600125), or NF-κB (Bay11-7082) (Figure 5D). To fur- moter activity through binding to its regulatory element
ther verify that NF-κB p65 is essential for ET-1-induced within the COX-2 promoter region, the wild-type (WT)
COX-2 expression, as shown in Figure 5E, transfection and mutated by a single-point mutation of the NF-κB
with p65 siRNA significantly reduced the p65 protein binding site (mt-κB) COX-2 promoters were constructed
expression (upper panel) and the ET-1-induced COX-2 (as illustrated in Figure 6E, upper panel). ET-1-
expression (lower panel). The results suggested that ET- stimulated COX-2 promoter activity was significantly
1-stimulated NF-κB translocation mediated through ET attenuated in bEnd.3 cells transfected with mt-κB-COX-
B
receptor, ERK1/2, p38 MAPK, and JNK1/2 is required 2 (Figure 6D, lower panel), indicating that NF-κB elem-
forCOX-2inductioninbEnd.3cells. ent was essential for ET-1-induced COX-2 promoter ac-
tivity. These results further confirmed that ET-1 induces
InvolvementofNF-κBinCOX-2genepromoteractivity COX-2 promoter activity via enhancing NF-κB binding
stimulatedbyET-1 to the κB binging site within COX-2 promoter region in
We have found that ET-1 stimulates translocation of bEnd.3 cells.
NF-κB p65 leading to COX-2 expression (Figure 5). We have found that ET-1 time-dependently induces
Next, we examined whether activation of NF-κB is PGE release (Figure 1D). Here, we further determined
2
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Figure5NF-κB(p65)isessentialforET1-1-inducedCOX-2expression.(A,B)Cellsweretreatedwith10nMET-1for6hintheabsenceor
presenceofBay11-7082.TheCOX-2proteinandmRNAexpressionweredeterminedbyWesternblotandRT-PCRasdescribedinFigure1.
(C)TimedependenceofET-1-stimulatedp65NF-κBtranslocationbysubcellularisolation,Westernblot,andimmunofluorescentstain.(D)Cells
werepretreatedwithU0126(1μM),SB202190(300nM),SP600125(300nM),BQ-788(1μM),orBay11-7082(10nM)for1handthenincubated
withET-1(10nM)for90min.ThenuclearfractionwasanalyzedbyWesternblot.(E)Cellsweretransfectedwithp65siRNAandthenexposedto
ET-1for6h.ThecelllysateswerecollectedandanalyzedbyWesternblottingusingananti-COX-2,anti-p65,anti-LaminA,oranti-GAPDH
(asaninternalcontrol)antibody.Dataareexpressedasmean±SEMofatleastthreeindividualexperiments(n=3ineachgroup;#P<0.01as
comparedwithET-1alone).
the involvement of these signaling components in ET-1- involved in inflammatory responses. The up-regulation
induced PGE release, as shown in Figure 6F, ET-1- of COX-2 has been shown to display a wide range of
2
induced PGE release was markedly attenuated by pre- biological activities in different tissues, including devel-
2
treatment with BQ-788, GPA2, GPA2A, U0126, opment, proliferation, cancers, and inflammation
SB202190, SP600125, Bay11-7082, or transfection with [14,15]. Moreover, ET-1 is elevated in the regions of vas-
p65 siRNA. These results demonstrated that ET - cular injuries andinflammation[7,8]. Circumstantialevi-
B
mediated activation of MAPKs (i.e., ERK, p38 MAPK, dence has further demonstrated that overexpression of
and JNK) and NF-κB by ET-1 is essential for COX-2 up- ET-1 on endothelial cells has deleterious effects on is-
regulationandPGE releaseinbEnd.3cells. chemic brain [1,5,6]. Reid et al. (1995) suggest that the
2
ET-1 model provides new insights into the mechanisms
Discussion of cerebral ischemia and reperfusion injury, and evalu-
Several lines of evidence have demonstrated that high ates the usefulness of novel strategies of neuroprotection
levels of PGs, synthesized by inducible COX-2, are [33]. ET-1 has been shown to up-regulate the expression
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Figure6ET-1-stimulatedCOX-2promoteractivityismediatedthroughNF-κB-dependentpathway.(A)TimedependenceofET-1-
enhancedNF-κBtranscriptionactivity,cellsweretransfectedwithaNF-κB-luciferasereportergeneandthenexposedtoET-1fortheindicated
times.(B)Aftertransfection,thecellswerepretreatedwithBQ-788(1μM),GPA2(1μM),GPA2A(1μM),U0126(1μM),SB202190(300nM),
SP600125(300nM),or(A)Bay11-7082(10nM)for1handthenincubatedwithET-1(10nM)for60min.(C)CellswerepretreatedwithBQ-788,
U0126,SB202190,orSP600125for1handthenincubatedwithET-1.Thep65NF-κBbindingactivitywasanalyzedbyChIP-PCRasdescribedin
Methods.(D)ForCOX-2promoteractivity,cellsweretransfectedwithaCOX-2-promoter-luciferasereportergeneandthenexposedtoET-1.After
transfection,thecellswerepretreatedwithBQ-788,GPA2,GPA2A,U0126,SB202190,SP600125,orBay11-7082for1handthenincubatedwith
ET-1for6h.(E)Schematicrepresentationofa50-promoterregionsofthemousedifferentCOX-2promoterconstructs,eitherwild-type(WT)or
mutationoftheκB-bindingsite(mt-κB)fusedtothepGL-luciferasereportergene,thetranslationalstartsite(+1)oftheluciferasereportergene
wasindicatedbyanarrow.CellsweretransfectedwithWTCOX-2promoterreportergene(WT-COX-2)orNF-κBmutatedCOX-2promoter
reportergene(mt-κB-COX-2)andthenincubatedwithorwithoutET-1for6h.Thepromoterreporterassaywasperformedasdescribedin
Methods.(F)CellswerepretreatedwithBQ-123,BQ-788,GPA2,GPA2A,U0126,SB202190,SP600125,Bay11-7082,ortransfectedwithp65siRNA
andthenincubatedwithET-1for6h.ThePGE levelswereanalyzedbyEIA.Dataareexpressedasmean±SEMofatleastthreeindividual
2
experiments(n=3ineachgroup;#P<0.01ascomparedwithET-1alone).
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of COX-2 through MAPKs in various cell types signaling pathways [24]. MAPKs activation by ET-1 has
[26,27,34]. However, little is known about the effect of been shown to modulate various cellular responses in
ET-1 on COX-2 expression in brain vascular endothelial several cell types [22,25]. Activation of ERK1/2 (p44/p42
cells. Here, we applied cultured models of mouse bEnd.3 MAPK) mightbe implicated in the expression of inflam-
cells coupled with Western blot analysis, selective matory genes in several models of vascular injury and
pharmacological inhibitors, transfection with siRNAs, inflammation [17,28]. In this study, we demonstrated
immunofluorescenct staining, and promoter assay to in- thatET-1stimulatedanET receptor-dependentcascade
B
vestigate the molecular mechanisms underlying ET-1- ofsequential ERK1/2phosphorylation (Figure 4E), which
induced COX-2 expression and PGE release. Our contributes to induction of COX-2 protein and mRNA
2
results demonstrate that in bEnd.3 cells, activation of levels (Figure 4A and 4B), promoter activity (Figure 6D),
ET receptor-dependent MAPKs (ERK1/2, p38, and and PGE release (Figure 6F).Theinvolvement ofERK1/
B 2
JNK1/2) and NF-κB signaling cascade is essential for 2 in COX-2 expression and PGE release was furthe
2
ET-1-inducedCOX-2geneexpressionandPGE release. confirmed by transfection of cells with p42 siRNA
2
ET-1activatesETreceptorsubtypes(ET andET )which (Figure 4D). These results are consistent with those of
A B
are coupled to various G proteins such as G and G and obtained with COX-2 expression induced by BK, throm-
q i
then lead to multiple signaling pathways and regulate di- bin,orET-1invariouscelltypes[17,26,28].Additionally,
verse cellular functions [7,20-22]. Thus, we first demon- we found that expression of COX-2 and release of PGE
2
strated a significant expression of ET receptor in mouse induced by ET-1 were also attenuated by the inhibitor of
B
bEnd.3cells(Figure2A).TheinvolvementofET receptors p38 MAPK or JNK1/2. Pretreatment with SB202190 or
B
in these responses is confirmed by that pretreatment with SP600125 both markedly reduced ET-1-induced ex-
BQ-788 (an ET receptor antagonist) reduced the ET-1- pression of COX-2 protein and mRNA (Figure 4A and
B
induced COX-2 protein and mRNA expression (Figure 2B 4B), promoter activity (Figure 6D), and PGE release
2
and 2C), promoter activity (Figure 6D), and PGE release (Figure 6F). Moreover, we also demonstrated that ET-1
2
(Figure6F),butnotbyanET receptorantagonistBQ-123. stimulates phosphorylation of p38 MAPK and JNK via
A
Subsequently, we confirmed these results by transfection anET -dependent manner(Figure4Cand4E).Similarly,
B
with ET siRNA (Figure 2D), suggesting that ET receptor we further confirmed these results by transfection with
B B
predominantly mediates ET-1-induced COX-2 expression siRNA for p38 MAPK or JNK1 that attenuated ET-1-
and PGE release in bEnd.3 cells. Next, several subtypes of induced COX-2 expression (Figure 4D). These data
2
GproteinsarepotentiallyimplicatedinET-1-inducedCOX- clearly indicated that in bEnd.3 cells, three MAPK cas-
2 expression. We use GPA2 (a G protein antagonist) and cades (i.e. ERK1/2, p38 MAPK, and JNK1/2) are
i
GPA2A(aG proteinantagonist)tointerruptGproteinsig- required for ET-1-induced COX-2 expression and
q
naling and consequent COX-2 expression (Figure 3A). PGE release. These results are consistent with those
2
Moreover, the inhibitory effects of GPA2 and GPA2A on of obtained with up-regulation of COX-2 by ET-1 via
COX-2inductionbyET-1werealso observedinitsmRNA p38 MAPK in glomerular mesangial cells or esophageal
(Figure3B),promoteractivity(Figure6D),andPGE release smooth muscle cells [27,34]. For the role of JNK1/2,
2
(Figure6F),indicatingthatET-1-inducedCOX-2expression we are the first presented that JNK1/2 plays a critical
and PGE release is mediated through a GPCR (i.e. ET ) role in induction of COX-2 by ET-1 in endothelial
2 B
coupling to either G or G protein in bEnd.3 cells,consist- (bEnd.3) cells.
i q
ent with previous studies from esophageal smooth muscle It has been well established that inflammatory responses
cells[34]andratbrainastrocytes[22].Incontrast,previous following exposure to extracellular stimuli are highly
reports have shown that ET-1 induces COX-2 expression dependent on activation of NF-κB transcription factor,
via ET receptors in peripheral lung microvascular smooth which plays an important role in regulation of several gene
A
muscle cells [25] and ET-1 (ET ) receptors linked to expression [40]. The 5’-flanking region of the COX-2 pro-
A
phospholipaseCandphospholipaseA activationandpros- moterhasbeenshowntocontainseveralbindingsequences
2
tanoidsecretion(e.g.PGE )inculturedhumanbrainmicro- for various transcription factors including NF-κB [41].
2
vascular endothelial cells [35,36]. However, in respiratory Therefore, the regulation of COX-2 transcription may be
andcardiovascularsystems,bothETreceptorsubtypes,ET mediatedbyaberrantactivationofseveraldistincttranscrip-
A
in particular, are involved in progression of several diseases tion factors dependent on agonists [29,42]. These reports
[37,38].Theredifferencesmaybeduetocelltypespecificor suggest that NF-κB plays a critical role in the regulation of
differentexperimentalconditions. COX-2 expression in the development of the inflammatory
Abnormal MAPK regulation might be implicated in responses.OurdatashowedthatET-1-inducedCOX-2gene
several models of CNS injury and inflammation [39]. expressionandPGE releasewassignificantlyabolishedbya
2
Several linesof evidence demonstrate that MAPKs could selective NF-κB inhibitor Bay11-7082 (Figures 5 and 6) or
be activated by GPCR agonists through different NF-κBp65siRNA(Figures5Eand6F),suggestingthatNF-
