Departments of Obstetrics and Gynecology and Physiology, Women and Children’s Health Research Institute, Cardiovascular Research Center and Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Alta. , Canada
Inflammatory changes in the vascular endothelium underlie the development of atherosclerosis, which leads to cardiovascular diseases such as myocardial infarctions and stroke, which are major causes of morbidity and mortality [1, 2] . Pre-menopausal women are relatively protected from cardiovascular diseases compared to men in the same age group. Higher circulating levels of the sex steroid estradiol (E 2 ) appear to mediate these anti-athero-genic effects [3, 4] . E 2 has been traditionally described as an anti-inflammatory factor contributing to the suppres-sion of endothelial inflammation, although the experi-mental evidence for such an effect is ambiguous [5, 6] . In
The sex hormone estradiol (E 2 ) appears to mediate both an-ti-atherogenic and pro-inflammatory effects in premeno-pausal women, suggesting a complex immunomodulatory role. Tumor necrosis factor (TNF) is a key pro-inflammatory cytokine involved in the pathogenesis of atherosclerosis and other inflammatory diseases. Alterations at the TNF recep-tors (TNFRs) and their downstream signaling/transcriptional pathways can affect inflammatory responses. Given this background, we hypothesized that chronic E 2 exposure would alter endothelial inflammatory response involving modulation at the levels of TNFRs and signaling pathways. HUVECs were used as the model system. Pre-treatment with E 2 did not significantly alter TNF-induced upregulation of pro-inflammatory molecules ICAM-1 (3–6 times) and VCAM-1 (5–7 times). However, pharmacological inhibition of tran-scriptional pathways suggested a partial shift from NF- � B (from 97 to 64%) towards the JNK/AP-1 pathway in ICAM-1 upregulation on E 2 treatment. In contrast, VCAM-1 expres-sion remained NF- � B dependent in both control ( � 96%) and
Received: February 2, 2012 Accepted after revision: August 13, 2012 Published online: $ $ $
Dr. Sandra T. Davidge 232 HMRC University of Alberta Edmonton, AB T6G 2S2 (Canada) E-Mail sandra.davidge @ ualberta.ca
Answers to Queries 1. Authors names and initials are correct 2. BAY 11-7085 - OK 3. Marham, Ont - inserted 4. ** p<0.01, inserted
Chakrabarti/Davidge
J Vasc Res2
contrast, pre-menopausal women are also at an increased risk for inflammatory and autoimmune disorders, such as rheumatoid arthritis and systemic lupus erythemato-sus [7–12] . These findings suggest an immunomodula-tory rather than purely anti-inflammatory role for E 2 on the female vasculature.
Tumor necrosis factor (TNF, also called TNF � ) is a 17-kDa pro-inflammatory cytokine involved in the patho-genesis of atherosclerosis and many other inflammatory diseases [13–15] . TNF induces upregulation of leukocyte adhesion molecules, such as intercellular (ICAM-1) and vascular cell adhesion molecules (VCAM-1), on the en-dothelium. Both ICAM-1 and VCAM-1 are expressed at low levels on the resting endothelium. Increased expres-sion of these adhesion molecules causes enhanced inter-actions with leukocytes, which are subsequently recruit-ed in a stepwise manner involving rolling, activation, firm adhesion and transmigration from the bloodstream into extravascular tissues, contributing to inflammatory effects [16–18] .
Despite the perceived anti-inflammatory properties of E 2 , its effects on inflammatory signaling in the endothe-lium are incompletely understood. A study by Caulin-Glaser et al. [19–21] has shown inhibitory effects of E 2 on interleukin (IL)-1-mediated adhesion molecule expres-sion, while others have suggested beneficial E 2 effects on endothelial apoptosis and cell migration [19–21] . In con-trast, studies from several other groups show equivocal or even aggravating effects of exogenous E 2 on TNF-me-diated changes [19, 21–24] . Most of these studies also dif-fer in the dose and duration of treatment with E 2 , adding further complexity to the inflammatory effects.
TNF exerts its biological actions through two different receptors, namely TNFR1 and TNFR2 [25] . Most of the pro-inflammatory effects of TNF are mediated through TNFR1, while the specific functions of TNFR2 are poor-ly understood [26–28] . Downstream of these receptors, TNF can activate various signaling and transcriptional pathways including nuclear factor (NF)- � B and mitogen-activated protein (MAP) kinase (e.g. c-Jun N-terminal ki-nase or JNK) which subsequently lead to upregulation of pro-inflammatory proteins. Most of these pathways are activated by TNFR1, while only Etk/BMX has been iden-tified as a specific downstream target of TNFR2 [29] . NF- � B is a major pro-inflammatory transcription factor in the endothelium that is activated by various pro-inflam-matory stimuli and contributes to the pathogenesis of atherosclerosis and other inflammatory diseases [30, 31] . In resting cells, the NF- � B components such as p65 and p50 remain in the cytoplasm in association with inhibitor
proteins known as inhibitory � B (I � B). Inflammatory events cause rapid phosphorylation and degradation of I � B, leading to the release of p65/p50 and their subse-quent migration into the cell nucleus, where they can bind to the promoter regions of pro-inflammatory genes to enhance gene transcription [reviewed in ref. 32 ]. The MAP kinase JNK is also activated through TNF. Active JNK can phosphorylate c-Jun, which in turn combines with c-Fos, migrates into the nucleus and promotes pro-inflammatory gene transcription [33] . Sp1 is another transcriptional factor commonly activated by E 2 , which is also a target of TNF action in the vasculature [34–36] .
Targeting TNFRs and the downstream signaling path-ways can affect the inflammatory process [37] . Yet little is known about the interactions between E 2 and TNF-activated signaling pathways in the vascular endotheli-um. Given this background, we hypothesized that chron-ic E 2 exposure would alter the endothelial inflammato-ry response involving modulation at the levels of both TNFRs and signaling pathways.
Methods
Reagents Dulbecco’s phosphate-buffered saline (PBS), M199 medium
with phenol red, porcine gelatin, NF- � B inhibitor BAY11-7085 (BAY), Sp1 inhibitor mithramycin A (MitA), the JNK/AP-1 inhib-itor SP600125 (SP) and cyclodextrin-encapsulated 17- � -E 2 were all bought from Sigma Chemical (St. Louis, Mo., USA). M199 medium without phenol red and fetal bovine serum (FBS) were obtained from Gibco/Invitrogen (Carlsbad, Calif., USA). Type 1 collagenase was purchased from Worthington Biochemical (Lakewood, N.J., USA). Triton X-100 and endothelial cell growth supplement were both from VWR International (West Chester, Pa., USA).
TNFR Agonists Highly selective TNFR agonists were a gift from the labora-
tory of Dr. Larry Guilbert, Department of Medical Microbiology and Immunology, University of Alberta. These peptides are TNF mutants (also called muteins) which selectively activate either TNFR1 (TNFR1 agonist, TNFR1A) or TNFR2 (TNFR2A). The specificity of these peptides has been validated both by Dr. Guil-bert’s group as well other research groups in different cell types, including endothelial cells [38, 39] .
Endothelial Cell Culture and Treatment HUVECs (human umbilical vein endothelial cells), a widely
used model for studying the vascular endothelium, were isolated from human umbilical cords obtained from the Royal Alexandra Hospital in Edmonton, Alta., Canada. The protocol was approved by the University of Alberta Ethics Committee and the investiga-tion also conformed to the principles outlined in the Declaration of Helsinki and also Title 45, US Code of Federal Regulations, Part 46, Protection of Human Subjects, effective December 13, 2001.
All subjects provided written informed consent before inclusion in the study. The umbilical vein was first flushed with PBS to re-move blood clots and then HUVECs were isolated using a type 1 collagenase-containing buffer. The cells were grown in a humidi-fied atmosphere at 37 ° C with 5% CO 2 /95% air in M199 medium with phenol red supplemented by 20% FBS as well as L -glutamine
(Gibco/Invitrogen), penicillin-streptomycin (Life Technologies) and 1% endothelial cell growth supplement. The endothelial na-ture of these cells was confirmed by staining for the endothelium-specific marker, von Willebrand’s factor (data not shown). Our laboratory has published extensively using HUVECs as a model for the vascular endothelium [40–44] .
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Fig. 1. E 2 effects on TNF-induced endothe-lial ICAM-1 expression. a HUVEC mono-layers were treated with/without E 2 (10 n M ) for 24 h prior to 4-hour stimulation with TNF (5 ng/ml). b , c HUVECs without ( b ) or with ( c ) E 2 (10 n M ) pre-treatment were washed and treated with NF- � B in-hibitor BAY (5 � M ), Sp1 inhibitor MitA (10 n M ) or JNK/AP-1 inhibitor SP (25 � M ) for 30 min prior to 4-hour stimulation with TNF (5 ng/ml). Cell lysates were analyzed by Western blotting for ICAM-1 and � -tu-bulin (loading control). Means 8 SEM from 4–5 independent experiments. * p ! 0.05 vs. untreated cells ( a ), and * p ! 0.05 and * * p ! 0.01 vs. TNF alone ( b , c ).
Once the HUVEC monolayers were 80–90% confluent, we in-cubated these in phenol red-free M199 medium (to prevent estro-genic actions of phenol red) with 1% FBS for 24 h with/without E 2 (10 n M ). Following E 2 treatment, the cells were washed once and stimulated with TNF (5 ng/ml).
Western Blotting Western blotting was performed on the HUVEC lysates as de-
scribed previously [44] . Bands for I � B � and I � B � (rabbit poly-clonal antibodies 9242 and 9248, respectively; Cell Signaling, Beverly, Mass., USA), VCAM-1 (rabbit polyclonal antibody sc-8304; Santa Cruz Biotechnologies, Santa Cruz, Calif., USA), ICAM-1 (mouse monoclonal antibody sc-8439; Santa Cruz Bio-technologies), TNFR1 (rabbit polyclonal antibody sc-7895; Santa Cruz Biotechnologies) and TNFR2 (rabbit polyclonal antibody ab15563; Abcam, Cambridge, Mass., USA) were normalized to � -tubulin (rabbit polyclonal antibody ab15246; Abcam). Bands for phospho-Etk (rabbit polyclonal antibody 3211; Cell Signaling) and phospho-c-Jun (rabbit polyclonal antibody 9164; Cell Signal-ing) were normalized to total Etk (mouse monoclonal antibody ABIN659597; Antibodies-Online GmbH, Atlanta, Ga., USA) and c-Jun (mouse monoclonal antibody 05-1076; Millipore, Temecula, Calif., USA), respectively. Anti- � -tubulin was used at 0.4 � g/ml, while all other antibodies were used at 1 � g/ml. Goat anti-rabbit and Donkey anti-mouse fluorochrome-conjugated secondary an-tibodies were purchased from LI-COR. The bands were detected by a LI-COR Odyssey BioImager and quantified by densitometry using corresponding software (LI-COR Biosciences, Lincoln, Nebr., USA). Samples prepared from a particular umbilical cord were run on the same gel. Cell lysates from untreated cells (no E 2 , no TNF) were loaded on every gel and the data were expressed as fold increase over the corresponding untreated control (i.e. no E 2 , no TNF).
Immunofluorescence HUVECs were fixed in 3% formalin and immunostained us-
ing overnight incubation with a rabbit polyclonal antibody against TNFR2 (rabbit polyclonal antibody ab15563; 1: 150; Abcam). Cells were then treated with AlexaFluor 488 (green)-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, Oreg., USA) for 30 min in the dark. Nuclei were stained with the Hoechst33342 nuclear dye from Molecular Probes. The cells were not permeabilized since TNFR2 exists on the cell surface associ-ated with the cell membrane. Cells were visualized under an Olympus IX81 fluorescent microscope (Carson Scientific Imag-ing Group; $ $ $ , Ont., Canada) using Slidebook 2D, 3D Timelapse Imaging Software (Intelligent Imaging Innovations, Denver, Colo., USA).
Statistics All data presented are means 8 SEM of 3–7 independent
experiments. Each independent experiment was performed on HUVECs isolated from a different umbilical cord. All data are expressed as fold change over the untreated control (no E 2 , no TNF). One-way analysis of variance (ANOVA) with an appropri-ate post hoc test was used to determine statistical significance, with an appropriate post hoc test (Dunnett’s test for comparison to control and Tukey’s test for multiple comparisons). To study the interaction between two different factors (such as E 2 and TNF), two-way ANOVA was used. A repeated-measure test was used
wherever applicable. The PRISM 5 statistical software (GraphPad Software, San Diego, Calif., USA) was employed for all analyses. p ! 0.05 was taken as significant.
Results
E 2 Alters the Transcriptional Pathways Mediating TNF-Induced ICAM-1 Expression We first examined the effects of 24-hour pre-treat-
ment with E 2 (10 n M ) on TNF-induced ICAM-1 expres-sion. We found that a 4-hour TNF stimulation signifi-cantly increased ICAM-1 protein levels (3- to 5-fold com-pared to the untreated control) in HUVECs, with no differences observed between the E 2 -treated and -un-treated cells ( fig. 1 a). E 2 alone had no effect on ICAM-1 levels. We also investigated the role of various transcrip-tional pathways on TNF-induced ICAM-1 expression. Specific pharmacological inhibitors were used to block these pathways, namely, NF- � B inhibitor BAY (5 � M ), Sp1 inhibitor MitA (10 n M ) or JNK/AP-1 inhibitor SP (25 � M ) that were all used for 30 min prior to a 4-hour stim-ulation with TNF. The concentrations of these inhibitors were determined based on previously published work by other research groups [45–47] . In the control (i.e. without E 2 ) cells, ICAM-1 upregulation was almost entirely de-pendent on NF- � B as this was completely ( � 97%) blocked in the presence of BAY ( fig. 1 b), similar to previously pub-lished reports [39, 48] . Surprisingly, in the E 2 -treated cells, NF- � B only appeared to play a partial ( � 64%) role, while inhibition of JNK (and hence, the AP-1 transcrip-tional pathway) by SP significantly ( � 91%) attenuated ICAM-1 protein levels ( fig. 1 c). Results with cyclodextrin (the vehicle for E 2 ) alone were not different from thosein E 2 -free cells (online suppl. fig. S1, for all suppl. mate-rial, see www.karger.com/doi/10.1159/000342736), while treatment with DMSO (the vehicle for the inhibitors used) had no effect by itself on TNF responses (online suppl. fig. S2). These data suggest E 2 -induced modula-tions in the TNF signaling machinery in endothelial cells, causing a shift from the NF- � B pathway towards the JNK/AP-1 pathway without affecting the amplitude of ICAM-1 upregulation.
E 2 Does Not Affect the Transcriptional Pathways Mediating TNF-Induced VCAM-1 Expression Next, we investigated the effects of 24-hour E 2 pre-
treatment on TNF-induced VCAM-1 protein levels. We found that E 2 treatment had no effects at all on TNF-me-diated VCAM-1 expression (5- to 7-fold compared to con-
trol; fig. 2 a), which remained almost wholly dependent on NF- � B signaling, both in the presence ( � 85%) or absence ( � 96%) of E 2 ( fig. 2 b, c). These results suggested a highly specialized modulation of the pro-inflammatory signal-ing machinery by E 2 , such that upregulation of VCAM-1 remained unchanged while regulation of ICAM-1 was al-tered.
E 2 Pre-Treatment Modulates TNF-Induced Degradation of I � B without Affecting the JNK Pathway Given the altered roles of NF- � B and JNK/AP-1 signal-
ing pathways in the E 2 -treated cells, we then proceeded to examine the TNF-mediated activation profiles of these pathways in the presence/absence of prior E 2 administra-
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Fig. 2. E 2 effects on TNF-induced endothe-lial VCAM-1 expression. a HUVEC mono-layers were treated with/without E 2 (10 n M ) for 24 h prior to 4-hour stimulation with TNF (5 ng/ml). b , c HUVECs without ( b ) or with ( c ) E 2 (10 n M ) pre-treatment were washed and treated with NF- � B in-hibitor BAY (5 � M ), Sp1 inhibitor MitA (10 n M ) or JNK/AP-1 inhibitor SP (25 � M ) for 30 min prior to 4-hour stimulation with TNF (5 ng/ml). Cell lysates were analyzed by Western blotting for VCAM-1 and � -tubulin (loading control). Means 8 SEM from 4–5 independent experiments. * * * p ! 0.001 vs. untreated cells ( a ), and * p ! 0.05 and * * p ! 0.01 vs. TNF alone( b , c ).
Fig. 3. E 2 effects on TNF-mediated NF- � B activation patterns. HUVEC monolayers were pre-treated with/without E 2 (10 n M ,24 h) before stimulation with TNF (5 ng/ml) for the indicated pe-riods of time. Cell lysates were analyzed by Western blotting for levels of I � B � ( a , c ), I � B � ( b , d ) and � -tubulin (loading control
for all panels). Means 8 SEM from 4–5 independent experiments. * p ! 0.05, * * p ! 0.01 and * * * p ! 0.001 vs. untreated control. I � B � and I � B � levels were also analyzed by two-way ANOVA ( e , f ) to examine specific effects of E 2 pre-treatment, TNF stimulation and the interactions (if any) between these.
tion. NF- � B activation is generally measured by the rapid degradation of intracellular I � B on the administration of a suitable stimulus [49, 50] . There are two main forms of I � B in the endothelium – namely I � B � and I � B � . I � B � is the form that is rapidly degraded by TNF, while the spe-cific role for I � B � is less clear [51, 52] . We found that I � B � was almost completely (90–91% decrease) degraded with-in 15 min of TNF treatment in both E 2 -treated and -un-treated cells. Even after 60 min, I � B � levels were only par-tially (38–54%) replenished, in agreement with previous work from our group [53] . In contrast, I � B � remained unaffected by TNF stimulation in the untreated cells, while it was partially degraded (42–48% decrease) on pro-longed TNF treatment (45 and 60 min) only in the E 2 -treated cells. Two-way ANOVA showed a significant effect of E 2 on I � B � responses to TNF. These data are presented in figure 3 and indicate a key regulatory role for chronic E 2 treatment on NF- � B activation.
We also examined the effects of E 2 on TNF-induced activation of JNK, measured by JNK phosphorylation levels. TNF caused a rapid (within 15 min) increase in phospho-JNK, which was reduced to basal levels within 45 min. In contrast to NF- � B, JNK phosphorylation pat-terns were identical in E 2 -treated and -untreated cells ( fig. 4 a, b, e). In addition, no effects of E 2 pre-treatment were observed on TNF-induced phosphorylation of c-Jun, a downstream target of JNK ( fig. 4 c, d, f). Interest-ingly, while the maximal response for JNK phosphoryla-tion was observed following 15 min of stimulation, the maximal phospho-c-Jun levels were seen at 30 min, which is consistent with c-Jun being downstream of JNK activa-tion. Collectively, these results suggest that the E 2 effects on TNF-treated HUVECs were not due to altered JNK activity but rather involved altered NF- � B signaling.
E 2 and TNF Differentially Regulate Expression Profiles of TNFR1 and TNFR2 We next studied the effects of E 2 on TNFR profiles.
Pro-inflammatory effects of TNF are generally mediated through TNFR1, while specific roles for TNFR2 are less clear [27] . Interestingly, neither E 2 pre-treatment nor 4-hour stimulation with TNF caused any statistically sig-nificant changes in protein levels of TNFR1 ( fig. 5 a). In contrast, TNFR2 levels were increased by E 2 treatment ( � 87% increase in Western blot), while two-way ANOVA showed significant interaction between E 2 and TNF on TNFR2 expression ( fig. 5 b). Similarly, E 2 pre-treatment (24 h) significantly increased ( � 60%) TNFR2 levels on the endothelial cell surface, as detected by immunofluo-rescence with an anti-TNFR2 antibody ( fig. 5 c), further
supporting the Western blot findings. These results sug-gest a potential role for TNFR2 in mediating E 2 -induced modulation of TNF signaling in the endothelium.
TNFR1 but Not TNFR2 Mediates EndothelialICAM-1 Upregulation In order to understand the roles of TNFR2 versus
TNFR1, we first examined the relative contributions of these receptors on endothelial ICAM-1 expression. In both E 2 -treated and -untreated cells, TNFR1A caused a highly significant (3- to 4-fold) increase in ICAM-1. TNFR2A alone had no effects at all. Combination of both TNFR1A and TNFR2A did not increase ICAM-1 levels above that observed with TNFR1A alone ( fig. 6 a). To determine if the TNFR2A peptide was functionally active, we examined Etk phosphorylation, a specific downstream target of TNFR2 [25, 54] . We found that TNFR2A was able to rap-idly phosphorylate Etk, suggesting that it was indeed acting through this receptor ( fig. 6 b). These findings indicate that endothelial ICAM-1 expression is largely dependent on TNFR1. While TNFR2 could potentially regulate the un-derlying signaling and transcriptional mechanisms, it did not alter the net effects of TNF signaling, i.e. the increase in ICAM-1 level. These results are also in accordance with the effects of E 2 treatment, where the total ICAM-1 levels remained unchanged ( fig. 1 a) while the transcriptional regulation was shifted from NF- � B to JNK/AP-1.
TNFR2 Is Critical for Alterations in TNF Signaling in the E 2 -Treated Endothelium Finally, we examined if TNFR2 signaling was indeed
involved in mediating E 2 -specific effects on TNF-stimu-lated endothelium, namely the degradation of I � B � and JNK-dependent ICAM-1 expression. On E 2- treated cells, TNFR1A alone did not significantly alter I � B � levels, while a combination of both TNFR1A and TNFR2Ainduced partial (35–46% decrease) I � B � degradation ( fig. 7 a), demonstrating the role of TNFR2 on this effect. In a similar vein, TNFR1A-mediated ICAM-1 expression (on E 2 -treated cells) was unaffected by JNK blockade; however, ICAM-1 expression in response to both receptor agonists was significantly attenuated ( � 45% decrease) by the JNK inhibitor ( fig. 7 b). Despite its modulatory roles on TNFR1 effects, activation of TNFR2 alone could nei-ther degrade I � B (online suppl. fig. S3) nor induce JNK activation (data not shown). These results demonstrate the requirement for TNFR2 signaling, together with TNFR1 effects, in inducing the E 2 -mediated changes in endothelial signaling both on NF- � B activity and ICAM-1 expression.
Fig. 4. E 2 effects on TNF-mediated JNK activation. HUVEC monolayers were pre-treated with/without E 2 (10 n M , 24 h) before stimulation with TNF (5 ng/ml) for the indicated periods of time. Cell lysates were analyzed by Western blotting for levels of phos-pho-JNK (p-JNK) and total JNK ( a , b ) or phospho-c-Jun (p-c-Jun)
and total c-Jun ( c , d ). Means 8 SEM from 5 independent experi-ments. * p ! 0.05 and * * * p ! 0.001 vs. untreated control. p-JNK/total JNK and p-c-Jun/total c-Jun levels were also analyzed by two-way ANOVA to examine specific effects of E 2 pre-treatment, TNF stimulation and the interaction (if any) between these ( e , f ).
Here we have shown the modulatory effects of chron-ic E 2 administration on TNF-mediated ICAM-1 upreg-ulation in the endothelium. E 2 increased the levels of TNFR2 but not TNFR1, and signaling through TNFR2 mediated a shift from the NF- � B to the AP-1 pathway on ICAM-1 expression. This shift in transcriptional regula-tion of ICAM-1 was accompanied by de novo degradation
of I � B � on TNF stimulation of the E 2 -treated cells. This work shows how E 2 regulates inflammatory processes whereby the underlying signaling mechanisms are al-tered.
TNF is a key pro-inflammatory cytokine involved in the pathogenesis of atherosclerosis and many other in-flammatory diseases. NF- � B activation is a major effect of TNF in the endothelium, which regulates functions such as inflammation and cell survival/apoptosis [39, 48,
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Fig. 5. Effects of E 2 and TNF on endothelial TNF receptor expression. a , b HUVEC monolayers were treated with/without E 2 (10 n M ) for 24 h prior to 4-hour stimulation with TNF (5 ng/ml). Cell lysates were Western blotted for TNFR1 ( a ) and TNFR2 ( b ) as well as for � -tubulin (loading control). Means 8 SEM from 6–7 inde-pendent experiments. Data were also analyzed by two-way ANOVA to examine specific effects of E 2 pre-treat-ment, TNF stimulation and the interactions (if any) between these. c HUVECs were treated with/without E 2 (10 n M ) for 24 h prior to fixation and immunostaining for TNFR2. Means 8 SEM from 3 independent experiments. * p ! 0.05 vs. untreated cells.
51, 55] . The intricacies of TNF signaling, the roles of spe-cific TNFRs and their regulation of NF- � B are still in-completely understood. For example, while upregulation of both ICAM-1 and VCAM-1 in TNF-treated endothe-lium is NF- � B dependent, there are clear differences in the regulation of these adhesion molecules. A study by May et al. [56] found that genistein, a tyrosine kinase in-hibitor with some estrogen-like activity, reduced VCAM-1 but not ICAM-1 expression in TNF-stimulated en-dothelial cells. The same study reported that sodiumorthovanadate, a protein phosphatase inhibitor, alsoreduced TNF-induced VCAM-1 expression while it fur-ther aggravated upregulation of ICAM-1. Similarly, genipin (a modulator of peroxisome proliferator-activat-ed receptor � ) has been shown to suppress TNF-induced VCAM-1 but not ICAM-1 expression via alteration of Akt and protein kinase C pathways [57] . Recently published work from our group has demonstrated that inhibition of neuronal nitric oxide synthase aggravates TNF-mediated upregulation of ICAM-1 and various pro-inflammatory cytokines, while levels of VCAM-1 and the anti-inflam-
matory cytokine IL-10 remain unaltered [58] . Collective-ly, these findings suggest that protein expression follow-ing the TNF-dependent activation of NF- � B is a complex phenomenon that can be differentially modulated by a variety of signaling pathways and external agents.
Within the cell, NF- � B is regulated by the specific in-hibitory proteins I � B � and I � B � . I � B � is known to be the main regulator of pro-inflammatory activity in re-sponse to diverse stimuli such as lipopolysaccharides, TNF and interferons. The regulatory effects of I � B � are less clear and variable [reviewed in ref. 32 ]. While most pro-inflammatory stimuli can induce degradation of I � B � , degradation of I � B � can occur only in a few in-stances [59–61] . Depending on the specific stimulus and the cell type, I � B � has been shown to be either synergis-tic with I � B � or acting quite differently [61, 62] . I � B � degradation occurs early in the inflammatory process, while that of I � B � is often delayed, a fact which may rep-resent a more persistent activation of NF- � B signaling in certain instances [63–65] . A study by Rao et al. [66] has recently shown that I � B � acts on a different set of genes
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Fig. 6. Selective activation of TNF receptors on endothelial ICAM-1 expression. a HUVEC monolayers were pre-treated with/with-out E 2 (10 n M , 24 h) before stimulation with TNFR-specific ago-nists (TNFR1A and TNFR2A, 10 ng/ml each) for 4 h. Cell lysates were analyzed by Western blotting for ICAM-1 and � -tubulin (loading control). b HUVEC monolayers were treated with the
selective TNFR2 agonist (TNFR2A, 10 ng/ml) for the indicated time periods. Cell lysates were analyzed by Western blotting for phospho-Etk (p-Etk) and total Etk. Means 8 SEM from 4–7 in-dependent experiments. * p ! 0.05 and * * p ! 0.01, vs. untreated control.
than I � B � , in addition to opposite effects on some of the same genes. Indeed, mice deficient in I � B � appear to be resistant to lipopolysaccharide-induced septic shock, suggesting a potential anti-inflammatory role for I � B � . Given this background, it is interesting to note that E 2 pre-treatment caused TNF-induced I � B � degradation, which was accompanied by a partial shift from NF- � B to AP-1 dependence on ICAM-1 expression.
Surprisingly, the shift to the JNK/AP-1 pathway on ICAM-1 expression did not involve any apparent changes to JNK activation. TNF effects on JNK activity (measured by phosphorylation of JNK and its downstream targetc-Jun) remained unchanged in both E 2 -treated and -un-treated cells, suggesting a degree of redundancy in the cytokine-activated inflammatory signaling pathways. It appears that in the presence of a robust NF- � B response (as in TNF stimulation in the absence of E 2 ), JNK is also activated but does not regulate ICAM-1. On the other
hand, when NF- � B activity is partially compromised (potentially due to I � B � degradation), the JNK/AP-1 pathway swings into action and contributes to ICAM-1 upregulation. These findings indicate that different TNF signaling mechanisms are involved in the presence of E 2 pre-treatment.
TNF signaling is involved in inflammation, oxidative stress and cell survival/apoptosis in the vascular endo-thelium. TNF responses are essential for wound healing and anti-bacterial defenses, while excessive and/or dys-regulated responses can contribute to inflammatory dis-eases [13] . TNF exerts its effects through two different receptors: TNFR1 and TNFR2. Traditionally, most of the pro-inflammatory and pro-apoptotic TNF effects are be-lieved to be dependent on TNFR1 [26] ; not surprisingly, we also found that TNFR1 was responsible for the up-regulation of the adhesion molecules in our system. How-ever, TNFR2 levels were increased in the E 2 -treated cells,
0
0.5
1.0
1.5I�
B�/�
-tub
ulin
(fol
d c
han
ge)
Untreate
dAlo
ne
TNFR1A: 45 m
in
TNFR1A: 60 m
in
*
Both: 4
5 min
**
Both: 6
0 min
�-Tubulin
I�B�
E2
a�-Tubulin
ICAM-1
b
0
2
4
6
ICA
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/�-t
ubul
in (f
old
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ang
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None – SP – SPUntreated
TNFR1A
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TNFR1A +TNFR2A
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Fig. 7. TNFR2 mediates E 2 effects in TNF-treated endothelium. a HUVEC monolayers were pre-treated with E 2 (10 n M ) for 24 h prior to being stimulated with TNFR-specific agonists (10 ng/ml each) for the indicated periods of time. Cell lysates were analyzed by Western blotting for I � B � and � -tubulin (loading control). Means 8 SEM from 5–7 independent experiments. * p ! 0.05 and * * p ! 0.01 vs. E 2 alone. b HUVEC monolayers were pre-treated
with E 2 (10 n M ) for 24 h prior to 30-min treatment with/with-out JNK/AP-1 inhibitor SP (25 � M ) followed by stimulation with TNFR-specific agonists (TNFR1A and TNFR2A, 10 ng/mleach) for 4 h. Cell lysates were analyzed by Western blotting for ICAM-1 and � -tubulin (loading control). Means 8 SEM from 5 independent experiments.
which were also decreased after 4 h of TNF stimulation, suggesting a role for TNFR2 in mediating at least some of the TNF effects in these cells.
TNFR1 signaling is relatively well characterized. On binding to TNF, TNFR1 associates with a number of in-tracellular proteins such as TRADD (TNFR-associated death domain) and TRAF2 (TNFR-associated factor 2), FADD (Fas-associated death domain protein) and RIP to form a signaling complex, which in turn activates I � B ki-nases leading to the phosphorylation and subsequent degradation of I � B molecules required for NF- � B activ-ity. The signaling complex soon dissociates from TNFR1 and then proceeds to activate a number of other pro-in-flammatory signaling cascades, including JNK phos-phorylation that leads to the activation of the AP-1 path-way [reviewed in ref. 32 ]. On the other hand, the signaling complexes formed by TNF-bound TNFR2 are less clearly known. To date, anti-apoptotic and angiogenic functions of TNFR2 have been determined in the endothelium, but its significance on inflammatory regulation is not clear [25, 28, 67, 68] . Our results suggest a novel regulatory role of TNFR2 in E 2 -treated cells, which allows for TNF-me-diated I � B � degradation and subsequent modulation of NF- � B activity. Alterations in NF- � B activity then allow the previously redundant AP-1 pathway to contribute to ICAM-1 upregulation. What is interesting is that TNFR2 activation in isolation could neither activate the said sig-naling pathways nor could it lead to ICAM-1 expression
[26] ) yet it profoundly affected these processes when act-ing in concert with TNFR1 signaling. The actual signal-ing proteins/complexes that are involved in mediating the TNFR2 effects remain to be determined.
Finally, it is not clear if the E 2 -induced increase in TNFR2 protein levels is either sufficient or critical for the alterations in TNF signaling observed in these cells. Since the rise in TNFR2 levels are quite modest, it is possible that the effects on NF- � B and JNK/AP-1 signaling might be more due to alterations in the components of TNFR2-associated signaling complexes rather than a simple in-crease in TNFR2 levels. Future research would be direct-ed to address these intriguing questions raised by our findings on E 2 -mediated alterations in TNFR2 signaling.
In conclusion, E 2 pre-treatment is shown to be a regu-lator of TNF effects in the vascular endothelium, which can help in our understanding of the sex-based differ-ences in inflammatory pathologies as well as the altered inflammatory state in pregnancy.
Acknowledgments
The study was supported by grants from the Heart and Stroke Foundation of Alberta, Northwest Territories and Nunavut and the Canadian Institutes for Health Research. S.C. is supported by a Canadian Institutes for Health Research fellowship and S.T.D. by Canada Research Chair in Women’s Cardiovascular Health funded by Alberta Innovates-Health Solutions as an Alberta Her-itage Foundation for Medical Research Scientist.
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