The role of caveolin-1 in PCB77-induced eNOS phosphorylation in human-derived
Eun Jin Lim1, Eric J. Smart2,4, Michal Toborek2,5, and Bernhard Hennig1,2,3
Molecular and Cell Nutrition Laboratory, College of Agriculture1, Graduate Centers for
Nutrition Sciences2, Toxicology3, and Departments of Pediatrics4and Neurosurgery5,
University of Kentucky, Lexington, KY, USA.
Running head: The role of caveolin-1 in PCB-induced endothelial activation
Address for correspondence:
Bernhard Hennig, Ph.D.
Molecular and Cell Nutrition Laboratory
University of Kentucky
Rm. 591 Wethington Health Sciences Bldg,
Lexington, KY 40536-0200, USA.
Page 1 of 31
Articles in PresS. Am J Physiol Heart Circ Physiol (October 12, 2007). doi:10.1152/ajpheart.00921.2007
Copyright © 2007 by the American Physiological Society.
PCBs may contribute to the pathology of atherosclerosis by activating
inflammatory responses in vascular endothelial cells. eNOS is co-localized with caveolae
and is a critical regulator of vascular homeostasis. PCBs may be proatherogenic by
causing dysfunctional eNOS signaling. The objective of this study was to investigate the
role of caveolin-1 in PCB-induced endothelial dysfunction with a focus on mechanisms
associated with eNOS signaling. Cells derived from an immortalized human vascular
endothelial cell line were treated with PCB77 to study nitrotyrosine formation through
eNOS signaling. Phosphorylation studies of eNOS, caveolin-1 and kinases, such as Src,
PI3K and Akt, were conducted in cells containing either functional or siRNA silenced
caveolin-1 protein. We also investigated caveolin-1-regulated mechanisms associated
with PCB-induced markers of peroxynitrite formation and DNA binding of NF-κB.
Cellular exposure to PCB77 increased eNOS phosphorylation and NO production, as well
as peroxynitrite levels. A subsequent PCB-induced increase in NF-κB DNA binding may
have implications in oxidative-stress mediated inflammatory mechanisms. The activation
of eNOS by PCB77 treatment was blocked by inhibitors of the Src/PI3K/AKT pathway.
PCB77 also increased phosphorylation of caveolin-1, indicating caveolae-dependent
endocytosis. Caveolin-1 silencing abolished both the PCB-stimulated AKT and eNOS
phosphorylation, suggesting a regulatory role of caveolae in PCB-induced eNOS
signaling. These findings suggest that PCB77 induces eNOS phosphorylation in
endothelial cells through a Src/PI3K/Akt-dependent mechanism, events regulated by
functional caveolin-1. Our data provide evidence that caveolae may play a critical role in
regulating vascular endothelial cell activation and toxicity induced by persistent
environmental pollutants such as coplanar PCBs.
Key words: PCB, atherosclerosis, endothelial activation, eNOS, caveolin-1, NF-κB
Page 2 of 31
From epidemiological studies, there is substantial evidence that cardiovascular
diseases are linked to environmental pollution. For example, increased hospitalization
rates have been reported for coronary heart disease in populations residing near areas
contaminated with persistent organic pollutants (45). Polychlorinated biphenyls (PCBs)
are widespread persistent environmental contaminants, which have been widely used for
various industrial applications. There is evidence linking the aryl hydrocarbon receptor
(AhR) with mechanisms associated with cardiovascular diseases (42) and that AhR
ligands such as coplanar PCBs may be atherogenic by disrupting the functions of
endothelial cells in blood vessels. We have demonstrated previously that coplanar as
well as non-coplanar PCBs can cause endothelial cell dysfunction as determined by
induction of inflammatory markers such as expression of cytokines and adhesion
molecules (15). Because the endothelium is in immediate contact with the blood,
endothelial cells are particularly susceptible to insult by circulating environmental
contaminants and their metabolites (16). The mechanisms by which PCBs induce
endothelial cell activation and dysfunction, oxidative stress and inflammation are not
fully understood. Oxidative stress-induced transcription factors, such as nuclear factor
κB (NF-κB), play a significant role in regulating inflammatory cytokine and adhesion
molecule production (6). Binding sites for NF-κB and related transcription factors were
identified in the promoter regions of a variety of inflammatory genes (20, 31) such as
interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1) or cyclooxygenase-2
(COX-2), all of which are upregulated during PCB toxicity (5, 14, 21, 49).
Endogenous nitric oxide (NO) is generated from arginine by a family of three
calmodulin-dependent NO synthase enzymes (3). NO produced from the endothelial
isoform eNOS regulates vital functions associated with vascular tone and local blood
flow (36). Abnormalities of NO production by vascular endothelial cells, and in
particular a decrease in bioavailable NO, can result in endothelial dysfunction and
accelerated atherosclerosis (18). Excessive production of NO can mediate cellular
toxicity by damaging critical metabolic enzymes and by reacting with superoxide to form
peroxynitrite, a potent oxidant. Thus, the beneficial protective effects of NO (e.g., anti-
inflammatory) are lost after reaction with superoxide anion (36). There are a number of
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kinases and phosphatases that can continuously associate or dissociate from the eNOS
signaling complex and thus provide a platform for regulatory processes (8). For example,
eNOS is activated by several growth factors through the PI3K/AKT pathways. (4, 9, 48,
50). Furthermore, caveolin-1 regulates NO signaling in the endothelium by binding to
and inhibiting eNOS (30). eNOS activity is regulated by Ca2+-calmodulin,
phosphorylation and interactions with caveolin-1 (26, 46). Activation of kinases such as
AKT can lead to eNOS activation and its dissociation from caveolin-1 (54). Thus,
regulation of eNOS by substrate and cofactor dependence, phosphorylation and
interaction with caveolin might all affect the level of bioavailable NO.
There is increasing evidence that membrane domains called caveolae play a
critical role in the pathology of atherosclerosis (23) and that the lack of the caveolin-1
gene may provide protection against the development of atherosclerosis (10). Caveolae
are particularly abundant in endothelial cells, where they are believed to play a major role
in the regulation of endothelial vesicular trafficking as well as the uptake of lipids and
related lipophilic compounds (28, 38), possibly including lipoprotein- and albumin-
associated persistent organic pollutants. Besides their possible role in cellular uptake of
lipophilic substances, caveolae contain an array of cell signaling molecules. In fact,
numerous genes involved in endothelial cell dysfunction, inflammation, and PCB toxicity
are associated with caveolae (11).
We and others have shown that persistent organic pollutants (such as PCBs) can
induce certain cell signaling pathways leading to the activation of proinflammatory
transcription factors such as NF-κB, which control inflammatory genes in endothelial
cells, including COX-2 and VCAM-1 (reviewed in (15). However, mechanisms, and in
particular intracellular signaling pathways, responsible for the regulation of PCB-
mediated endothelial cell activation are not well understood. In the present investigation,
we hypothesized that caveolae play a critical role in endothelial activation and associated
vascular toxicity induced by persistent environmental pollutants such as PCBs. Our data
suggest that PCB77 induces proinflammatory parameters through eNOS signaling and
that caveolae may play a critical role in regulating vascular endothelial cell activations
and toxicity induced by persistent environmental pollutants such as PCB77.
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Materials and Methods
Anti-caveolin-1 antibody was obtained from Affinity BioReagents (Golden, MO).
Anti-phospho-caveolin (Tyr-14), anti-eNOS, anti-phospho-eNOS (Ser-1177), anti-AKT,
anti-phospho-AKT (Ser-473), and anti-rabbit Ig horseradish peroxidase linked antibodies
were purchased from Cell Signaling Technology (Danvers, MA). Anti-p65 NF-κB
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin
was purchased from Sigma (Saint Louis, MO). Supplies and reagents for SDS-PAGE
were purchased from Bio-Rad (Hercules, CA). Both the PI3K inhibitors (Ly294002 and
wortmannin) and the Src inhibitor (PP2) were purchased from Calbiochem (San Diego,
CA). 3, 3, 4, 4’ -tetrachlorobiphenyl (PCB77) was purchased from AccuStandard (New
Primary endothelial cells were isolated from porcine pulmonary arteries and
cultured as previously described (17). EAhy926 cells (a kind gift from Dr. C. S. Edgell,
University of North Carolina) were maintained in Dulbecco’ s modified Eagle’ s medium
(DMEM) (Invitrogen, Carlsbad, California) supplemented with 10% fetal bovine serum
(FBS; Hyclone, Logan, UT) and antibiotics. Cell cultures were grown until confluent,
and then synchronized by maintaining in 1% serum for 16 hr before treatment for various
The EA.hy926 line was derived by fusing human umbilical vein endothelial cells
with the permanent human cell line A549 (7). EA.hy926 cells are contact inhibited in
growth, show reduced growth factor requirements, express von Willebrand factor and
upregulate ICAM-1, VCAM-1 and E-selectin expression upon stimulation with TNF-
alpha (2). EA.hy926 cells were used as a model for endothelial cells because, like
endothelial cells, EA.hy926 cells endogenously express eNOS and caveolin-1.
Caveolin-1 siRNA and transfection
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Caveolin-1 gene silencer was designed as previously described (37). Cells were
transfected with control siRNA or caveolin-1 siRNA at a final concentration of 80 nM
using GeneSilencer (Genlantis, San Diego, CA) in Optimem I medium (Invitrogen,
Carlsbad, California). Cells were incubated with transfection mixtures for 4 hr and then
replaced with regular medium. Forty-eight hr after transfection, cells were synchronized
overnight and then treated with PCB77 (2.5 µM) or vehicle.
Cells were treated with either vehicle (0.1% DMSO), PCB77 (2.5 µM), PI3K
inhibitors (Ly294002 and wortmannin), or Src inhibitor (PP2) in DMEM with 1% FBS.
Cells were washed twice, scraped in ice-cold PBS and centrifuged. Appropriate amounts
of boiling lysis buffer (1% SDS, 1 mM sodium orthovandate, 10 mM Tris, pH 7.4) were
added to the cell pellets. The samples were boiled for 5 min and passed several times
through a 26-gauge needle. After centrifugation, protein concentrations of supernatants
were determined using BCA protein assay reagent (Pierce, Rockford, IL). Cellular
proteins were resolved by SDS-PAGE (12-15% acrylamide) and transferred to
nitrocellulose membranes. Blots were incubated overnight in TBST (Tris-buffered saline,
pH 7.6, containing 0.05% Tween 20) containing 5% milk powder. After three washes
with TBST, membranes were incubated overnight with the primary antibody (~1,000-fold
diluted in TBST containing 5% bovine serum albumin) and for 1 hr with HRP-conjugated
secondary antibody (~5,000-fold diluted). Bound antibodies were detected using an ECL
(Amersham Life Science, Birmingham, UK).
Measurement of total NO
The accumulation of nitrite and nitrate (total NO) in culture media after PCB77
treatment was measured as described previously (32). The cell culture supernatants were
centrifuged at 7,740 x g with a micropore filter (Ultrafree-MC microcentrifuge device,
UFC3; Millipore) to remove substances larger than 10 kDa. The filtrates were analyzed
by using a Nitric Oxide Assay kit (R&D Systems, Minneapolis, MN) according to the
manufacturer’ s protocol.
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Immunoprecipitation of nitrotyrosine was carried out using anti-nitrotyrosine
antibody (Cayman, Ann Arbor, MI) as described previously (40). Briefly, for
nitrotyrosine immunoprecipitation, samples were incubated with Protein A/G Plus-
agarose beads (Santa Cruz Biotechnology) and incubated overnight at 4oC with anti-
nitrotyrosine antibody. The beads were recovered by centrifugation at 1500 x g for 5 min
at 4°C and washed four times with 1 ml of PBS. Bound proteins were eluted by boiling
in loading buffer. Immunoprecipitates were separated by 7.5% SDS-PAGE and blotted
onto a nitrocellulose membrane. After blocking with 5% milk, the membrane was
incubated for 2 hr with mouse monoclonal anti-nitrotyrosine antibody in a 1:1,000
dilution. The bands were visualized by incubation of membranes with HRP-conjugated
secondary antibody followed by ECL (Amersham Life Science, Birmingham, UK).
Electrophoretic mobility shift assays
Nuclear extracts from EA.hy926 cells were prepared as described previously (24).
Synthetic 5'-biotinylated complementary oligonucleotides were purchased from
Integrated DNA Technologies (Coralville, IA). Nuclear extracts were incubated for 20
min with biotin-labeled oligonucleotide probes containing the enhancer DNA element for
NF- B (5'- AGTTGAGGGGACTTTCCCAGGC -3'). Gel mobility shift assay was
carried out using a LightShiftTM chemiluminescent EMSA kit (Pierce, Rockford, IL) (41).
Images were quantified, and data were analyzed using the Scion Image and Sigma
Stat software, respectively. Comparisons between treatments were made by one- or two-
way ANOVA with post-hoc comparisons of the means made by Tukey’ s tests. A
statistical probability of p< 0.05 was considered significant.
PCB77 increases eNOS phosphorylation and NO production in endothelial cells.
eNOS is an enzyme that generates NO and is activated by phosphorylation of a
serine residue at position 1177 in endothelial cells. eNOS activation was tested in both
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primary porcine endothelial cells and in EA.hy926 cells. Both cell types were incubated
with PCB77 for 5, 10 or 30 min. PCB77 markedly induced eNOS phosphorylation
between 5 and 10 min in both cell types (Figs. 1A and 1B). In EA.hy926 cells,
densitometric analysis of phospho-eNOS bands showed that PCB77 treatment increased
eNOS phosphorylation by 7-fold after 5 min and 4-fold after 10 min (Fig. 1B).
To investigate whether PCB77-induced eNOS phosphorylation results in an
increase in NO production and may be involved in PCB77-induced endothelial
dysfunction, we measured NO production and nitrotyrosine formation. Treatment with
PCB77 rapidly increased NO production, with close to a 3-fold increase as early as 5 min
after exposure. EA.hy926 cell-derived NO returned to near control levels after 10 min of
PCB exposure (Fig. 1C). This increase in NO production was followed by a marked
increase in nitrotyrosine formation 30 min after PCB77 treatment (Fig. 1D).
PCB77 induces eNOS phosphorylation through a Src/PI3K/AKT-dependent
Several protein kinases, including the serine threonine kinase AKT (PKB), have
been shown to be localized upstream of eNOS and can phosphorylate eNOS. Therefore,
the effect of PCB77 on AKT phosphorylation was investigated. PCB77 caused maximal
AKT Ser-473 phosphorylation 5 min after PCB exposure (Fig. 2A).
To investigate whether PI3K is involved in PCB77-induced eNOS
phosphorylation, cells were preincubated for 2 hr with the PI3K inhibitors LY294002 or
wortmannin. Both of these PI3K inhibitors decreased PCB77-stimulated AKT and eNOS
activation while not affecting total protein levels (Fig. 2B). These data indicate that PI3K
is critical for PCB77-induced serine 1177 eNOS. Src family tyrosine kinases have been
known to be localized upstream of PI3K/AKT pathway (19). To test if Src can also
phosphorylate eNOS via the PI3K/AKT pathway, we determined the effect of the Src
inhibitor PP2 on eNOS activation by PCB77. EA.hy926 cells were preincubated with or
without PP2 for 2 hr prior to PCB77 stimulation. PP2 significantly decreased eNOS
phosphorylation (Fig. 2C). These results suggest that PCB77 stimulates Src which in
turn can activate the PI3K/AKT pathway to ultimately lead to eNOS phosphorylation.
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PCB77 increases NF-κ κB DNA binding through the Src/PI3K/AKT pathway.
NF-κB is a critical oxidative stress sensitive transcription factor, which regulates
inflammatory genes in endothelial cells. Therefore, we tested whether the Src/PI3K/AKT
pathway can also be involved in PCB77-induced activation of NF-κB DNA binding. As
shown in Figure 3, treatment with PCB77 for 1 hr markedly induced NF-κB activation.
Both the Src inhibitor PP2 and the PI3K inhibitors LY294002 and wortmannin decreased
the PCB-induced activation of NF-κB, suggesting a regulatory involvement of the
Src/PI3K/AKT pathway (Fig. 3A). The specificity of NF-κB binding to the consensus
oligonucleotide was demonstrated by a decrease of the NF-κB band by adding an excess
of unlabeled oligonucleotide and antibody against the p65 subunit of NF-κB (Fig. 3B).
PCB77 induces caveolin-1 phosphorylation.
It has been suggested that phosphorylation of caveolin-1 is required for the
engagement of the signaling machinery responsible for caveolae-mediated endocytosis
(47). To examine whether PCB77 can induce phosphorylation of caveolin-1, cells were
incubated with PCB77 for up to 10 min. PCB77 markedly induced caveolin-1
phosphorylation at 5 min and then returned to near control levels by 10 min (Fig. 4).
Caveolin-1 silencing reduces PCB-induced eNOS and AKT phosphorylation.
To determine the role of caveolin-1 in PCB77-dependent phosphorylation of
eNOS, we utilized small interfering RNA to specifically silence caveolin-1. Caveolin-1
siRNA reduced caveolin-1 expression by ~80 % as compared with control cells (Fig. 5A).
However, β-actin and total protein levels of eNOS, and AKT were unchanged in
caveolin-1 siRNA-transfected cells, indicating the specificity of the silencing (Figs. 5A,
5B and 5C). Subsequently, we measured the effect of PCB77 on phosphorylation of
eNOS and AKT in caveolin-1 silenced EA.hy926 cells. Caveolin-1 silencing
significantly decreased the PCB77-induced phosphorylation of eNOS Ser-1177 (Fig. 5B)
and AKT Ser-473 (Fig 5C). Caveolin-1 silencing did not affect AKT phosphorylation
and expression in cells without PCB treatment (Figure 5C). Similar results were
observed with the eNOS data (Figure 5B).
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Caveolin-1 silencing reduces the activation of NF-κ κB by PCB77.
To determine the role of caveolin-1 in PCB77-dependent NF-κB activation,
siRNA technology was utilized to specifically silence caveolin-1. Similar to the
experiments presented in Figure 5, caveolin-1 siRNA reduced caveolin-1 expression by
~80 % as compared with control cells without affecting β-actin levels. As illustrated,
caveolin-1 silencing significantly decreased the PCB77-induced the activation of NF-κB
There is substantial evidence that the etiology of cardiovascular diseases is linked
to exposure of environmental pollution. For example, the mortality from cardiovascular
diseases was increased among Swedish capacitor manufacturing workers exposed to
PCBs for at least five years (12). Also, in workers exposed to phenoxy herbicides and
PCBs in waste transformer oil, deaths were due primarily to complications from
cardiovascular disease (13). Most importantly, a recent study reported increased
hospitalization rates for coronary heart disease in populations residing near areas
contaminated with persistent organic pollutants (45). Many environmental pollutants,
including coplanar PCBs such as PCB77, interact with the AhR to initiate xenobiotic
metabolizing activity linked to an increase in cellular oxidative stress (1). It has been
suggested that AhR activation is a critical participant in mechanisms associated with
cardiovascular diseases (42, 53) and that AhR ligands may be atherogenic by disrupting
the functions of endothelial cells in blood vessels.
The vascular endothelium serves a critical role in the regulation of both the
structure and function of blood vessels. Endothelial cells not only form a barrier
protecting the underlying vascular tissue, but these cells also generate signaling
molecules, which serve diverse autocrine and paracrine functions. Endothelial cell
activation and dysfunction and subsequent inflammatory events are considered critical in
the etiology of vascular diseases such as atherosclerosis (39). Environmental toxicants,
once absorbed, distribute themselves to tissues, especially adipose, where they are in
dynamic equilibrium with the blood. Thus, risk factors of such pollutants such as PCBs
are chronic and can continuously amplify pathologies of diseases that are associated with
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endothelial dysfunction. Data from our present study confirm that endothelial exposure
to PCB77 provides a prooxidative cellular environment sufficient to induce oxidative
stress-sensitive transcription factors such as NF-κB.
We have shown previously that coplanar PCBs cause endothelial cell dysfunction,
which was associated with an increase in activity of cytochrome P4501A and cellular
oxidative stress (52). Subsequent in vitro and in vivo studies demonstrated a strong link
between exposure to PCBs and induction of inflammatory markers characteristic of early
events in the pathology of atherosclerosis (14). Using mice lacking the AhR gene, we
also showed that the PCB-induced proinflammatory properties were dependent on an
intact AhR (14).
Mechanisms of PCB-induced oxidative stress and inflammation are not clear;
however, eNOS dysfunction resulting in peroxynitrite formation and protein tyrosine
nitration has been proposed to be an independent marker of cardiovascular disease (35).
In our current study we demonstrated that exposure to PCB77 can increase
phosphorylation of eNOS, with a subsequent increase in peroxynitrite formation and
induction of NF-κB. There is evidence that coplanar PCBs can markedly stimulate
reactive oxygen species production through uncoupling of cytochrome P4501A (43).
These data and results from our own work (14, 40) strongly support our hypothesis that
exposure to coplanar PCBs can lead to endothelial dysfunction and induction of
inflammatory markers through eNOS signaling. In the current study we demonstrated
that exposure to PCB can increase phosphorylation of eNOS in 5/10 min, with a
subsequent increase in peroxynitrite formation at 30 min and induction of NF-κB at 1 h.
We also present evidence that kinase signaling pathways upstream of eNOS may
be critical regulatory mechanisms involved in the toxicity of persistent organic pollutants
such as PCBs. Using appropriate inhibitors, we were able to demonstrate that PCB77 can
induce eNOS phosphorylation in endothelial cells through a Src/PI3K/AKT-dependent
signaling pathway. Our data are supported by mechanistic studies of AhR activation by
dioxin (TCDD), where it was observed that TCDD was able to directly activate c-Src
tyrosine kinase (34). Activated Src can recruit the p85 subunit of phosphatidylinositol 3-
kinase, resulting in PI3K/AKT activation (19).
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There is evidence that caveolae play a critical role in the pathology of
atherosclerosis and that the lack of the caveolin-1 gene reduces an atherogenic outcome
(44). Caveolae compartmentalize signal transduction molecules which regulate multiple
endothelial functions. For example, the mechanisms underlying eNOS localization in
caveolae and associated activity have been studied extensively (29). For optimal
activation, eNOS is targeted to caveolae, and caveolin-1, the major coat protein of
caveolae, regulates eNOS activity (27). We provide novel data, demonstrating that
silencing of the caveolin-1 gene can markedly down-regulate PCB-induced eNOS
phosphorylation, peroxynitrite formation and activation of NF-κB. Our data strongly
support our hypothesis that caveolae present a critical platform in regulating
inflammatory signaling pathways induced by environmental pollutants such as PCBs. As
mentioned above, an important mechanism that is regulated by caveolae is eNOS
signaling, and cross-talk between caveolin-1 and eNOS may be an important factor in
mechanisms of the pathology of atherosclerosis. In human blood vessels, eNOS was
significantly increased in the endothelium overlying intimal thickening and
atherosclerotic plaques compared with the adjacent endothelium overlying normal media
(55). Interestingly, the endothelial caveoliln-1 to eNOS ratio increased 5-fold only in
endothelium overlying plaque, suggesting a close relationship between increased
caveolin-1 and atherosclerotic vessels (55).
Caveolae may also serve as critical transport vesicles involved in transcytosis of
solutes, membrane components, proteins, viruses, extracellular ligands (22) and possibly
lipophilic compounds such as PCBs. Thus, in addition to serving as a critical platform
during inflammatory signaling, caveolae also may facilitate and regulate cellular entry of
PCBs. It has been shown that PCBs in plasma are associated primarily with albumin and
low-density lipoproteins (LDL) (33). Since the albumin acceptor gp60 (51) as well as the
lipid/lipoprotein receptor CD36 (25) are enriched in caveolae, membrane caveolae likely
mediate the first contact of endothelial cells with PCBs suggesting caveolae may have
implications in PCB toxicity.
In summary, we have described a new mechanism of PCB77-induced activation
of endothelial cells that involves a rapid PI3K-dependent activation of AKT and
subsequent serine phosphorylation of eNOS. The mechanisms by which PCB77 induces
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PI3K-AKT-eNOS activation appear to be regulated by and dependent on functional
caveolin-1 (Figure 7). Our data provide an understanding of how cross-talk between
caveolin-1 and eNOS signaling can lead to PCB77-mediated induction of
proinflammatory genes in vascular endothelial cells. These mechanisms include eNOS as
a potential source of reactive oxygen species and peroxynitrite. Because caveolae and
caveolins have been implicated in several human diseases and in particular vascular
diseases, our data may have implications in understanding mechanisms of inflammatory
diseases induced by exposure to environmental pollutants.
This research was supported by grants from NIEHS, NIH (P42ES07380) and the
University of Kentucky AES.
Page 13 of 31
Figure 1. PCB77 stimulates eNOS phosphorylation and NO production in endothelial
cells. Both primary porcine endothelial cells (Figure 1A) and EA.hy926 cells (Figure
1B) were incubated with 2.5 µM PCB77 for the indicated times. eNOS phosphorylation
was determined by western blotting with an antibody directed against phospho-eNOS (p-
eNOS) and total eNOS. Densitometry results shown in parallel represent mean ± SEM of
three independent experiments. NO production (Figure1C) is represented as mean ±
SEM of three independent experiments. Figure 1D represents immunodetection of
nitrotyrosine after treatment with PCB77 (2.5 µM) for 30 min. The blot is a
representative of three independent experiments. *Significantly different compared to
Figure 2. PCB77 induces eNOS phosphorylation through a Src/PI3K/AKT-dependent
mechanism. Cells were treated with PCB77 (2.5 µM) for the indicated times and
phosphorylation of AKT was determined by western blot analysis using anti-p-AKT
antibody (Figure 2A). Cells were treated with PCB77 in the absence or presence of PI3K
inhibitors (wortmannin, 200 nM or Ly294002, 50 µM; 2 hr pretreatment).
Phosphorylation of AKT and eNOS was determined by western blotting using specific
antibodies (Figure 2B). To test if Src also can phosphorylate eNOS, cells were treated
with PCB77 for 5 min with or without the Src inhibitor (PP2, 1 µM; 2 hr preincubation.
Total cell lysates were subjected to western blot analysis (Figure 2C). Densitometry
results shown in parallel represent means ± SEM of three independent experiments.
*Significantly different compared to vehicle control. **Significantly different compared
Figure 3. PCB77 increases NF-κB DNA binding through the Src/PK3K/AKT pathway.
EA.hy926 cells were treated with PCB77 (2.5 µM) for 2 hr in the absence or presence of
PI3K inhibitors (wortmannin, 200 nM or Ly294002, 50 µM; 2 hr preincubation) or the
Src inhibitor (PP2, 1 µM; 2 hr preincubation). NF-κB binding was determined by
electrophoretic mobility shift assay with nuclear proteins extracted from treated cells.
Page 14 of 31
NF-κB DNA binding complexes are identified by arrows. Binding intensity was
quantified by densitometry. Densitometry results shown in parallel represent mean ±
SEM of three independent experiments (Figure 3A). *Significantly different compared to
vehicle control. **Significantly different compared to PCB77. Gel shift assay using p65
antibody, and competition assay were used to confirm NF-κB-DNA binding activity.
Lane 1, free probe; Lane 2, nuclear extract; Lane 3, nuclear extract plus 200 x excess of
unlabeled NF-κB probe; Lane 4, nuclear extract plus anti-p65 antibody (Figure 3B).
Figure 4. PCB77 induces caveolin-1 phosphorylation. EA.hy926 cells were incubated
with 2.5 µM PCB77 for the indicated times. Total cell lysates were probed with anti-
phospho-Cav-1(p-Cav-1), and anti-caveolin-1 (Cav-1). Each blot is a representative of
three independent experiments. Densitometry results shown in parallel represent mean ±
SEM of three independent experiments. *Significantly different compared to vehicle
Figure 5. Caveolin-1 silencing reduces PCB-induced eNOS and AKT phosphorylation.
Cells were transfected with siRNA for caveolin-1 (Cav-1 siRNA) or with control siRNA
(Ctr- siRNA) and treated with PCB77 (2.5 µM) for the indicated times. Cell lysates were
probed with Cav-1and β-actin antibodies (Figure 5A) or with anti-p-eNOS, anti-eNOS
(Figure 5B), anti-p-AKT, and anti-AKT (Figure 5C). Densitometry results shown in
parallel represent mean ± SEM of three independent experiments. *Significant different
compared to vehicle control. **Significant different compared to PCB77.
Figure 6. Caveolin-1 silencing reduces the activation of NF-κB by PCB77. Cells were
transfected with siRNA for caveolin-1 or with control siRNA (Cav-1 siRNA or Ctr-
siRNA, respectively) and treated with PCB77 (2.5 µM) for 5 min. The efficiency and
specificity of silencing were determined by western blotting with Cav-1and β-actin
antibodies. Electrophoretic mobility shift assay for NF-κB was performed with nuclear
proteins extracted from endothelial cells. NF-κB DNA binding complexes are identified
by arrows. Binding intensity was quantified by densitometry. Densitometry results
Page 15 of 31
shown in parallel represent mean ± SEM of three independent experiments. *Significant
different compared to vehicle control. **Significant different compared to PCB77.
Figure 7. Proposed model for the mechanism of PCB77-mediated EA.hy926 cell
activation. PCB77 treatment activates phosphorylation of eNOS via the Src-PI3K-AKT
pathway, followed by an increase in NO production. This process is regulated by
caveolin-1 phosphorylation. In addition, treatment with PCB77 can induce the formation
of superoxide radicals via uncoupling cytochrome P450 1A1 (43). Under oxidative stress,
the reaction between superoxide radicals and NO is facilitated, resulting in peroxynitrite
formation. Peroxynitrite, being a potent oxidative and nitrating agent, can activate NF-
κB and induce the expression of adhesion molecules in endothelial cells. eNOS is
activated by PI3K-AKT signaling and Cav-1 phosphorylation, leading to increased
production of NO. Caveolin-1 is necessary for eNOS phosphorylation by PCB77.
Page 16 of 31
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142x180mm (300 x 300 DPI)
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211x145mm (300 x 300 DPI)
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155x156mm (300 x 300 DPI)
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189x177mm (300 x 300 DPI)
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179x168mm (300 x 300 DPI)
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230x152mm (300 x 300 DPI)
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158x146mm (300 x 300 DPI)
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160x167mm (300 x 300 DPI)
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165x161mm (300 x 300 DPI)
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156x165mm (300 x 300 DPI)
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211x156mm (200 x 200 DPI)
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