Research Paper Received: February 2, 2012 Accepted after revision: August 13, 2012 Published online: $ $ $

J Vasc Res DOI: 10.1159/000342736

Estradiol Modulates Tumor Necrosis Factor-Induced Endothelial Inflammation: Role of Tumor Necrosis Factor Receptor 2 Subhadeep Chakrabarti Sandra T. Davidge 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

Key Words Adhesion molecules ⴢ Estradiol ⴢ HUVEC ⴢ TNFR1 ⴢ TNFR2

Abstract The sex hormone estradiol (E2) appears to mediate both anti-atherogenic and pro-inflammatory effects in premenopausal 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 receptors (TNFRs) and their downstream signaling/transcriptional pathways can affect inflammatory responses. Given this background, we hypothesized that chronic E2 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 E2 did not significantly alter TNF-induced upregulation of pro-inflammatory molecules ICAM-1 (3–6 times) and VCAM1 (5–7 times). However, pharmacological inhibition of transcriptional pathways suggested a partial shift from NF-B (from 97 to 64%) towards the JNK/AP-1 pathway in ICAM-1 upregulation on E2 treatment. In contrast, VCAM-1 expression remained NF-B dependent in both control (⬃96%) and

© 2012 S. Karger AG, Basel 1018–1172/12/0000–0000$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

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Accessible online at: www.karger.com/jvr

E2 treated (⬃85%) cells. The pro-inflammatory TNF effects were mediated by TNFR1. Interestingly, E2 pre-treatment increased TNFR2 levels in these cells. Concomitant TNFR2 activation (but not TNFR1 activation alone) led to the shift towards JNK/AP-1-mediated ICAM-1 upregulation in E2-treated cells, suggesting the effects of chronic E2 to be dependent on TNFR2 signaling. Copyright © 2012 S. Karger AG, Basel

Introduction

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 (E2) appear to mediate these anti-atherogenic effects [3, 4]. E2 has been traditionally described as an anti-inflammatory factor contributing to the suppression of endothelial inflammation, although the experimental evidence for such an effect is ambiguous [5, 6]. In Dr. Sandra T. Davidge 232 HMRC University of Alberta Edmonton, AB T6G 2S2 (Canada) E-Mail sandra.davidge @ ualberta.ca

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contrast, pre-menopausal women are also at an increased risk for inflammatory and autoimmune disorders, such as rheumatoid arthritis and systemic lupus erythematosus [7–12]. These findings suggest an immunomodulatory rather than purely anti-inflammatory role for E2 on the female vasculature. Tumor necrosis factor (TNF, also called TNF) is a 17kDa pro-inflammatory cytokine involved in the pathogenesis 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 endothelium. Both ICAM-1 and VCAM-1 are expressed at low levels on the resting endothelium. Increased expression of these adhesion molecules causes enhanced interactions with leukocytes, which are subsequently recruited 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 E2, its effects on inflammatory signaling in the endothelium are incompletely understood. A study by CaulinGlaser et al. [19–21] has shown inhibitory effects of E2 on interleukin (IL)-1-mediated adhesion molecule expression, while others have suggested beneficial E2 effects on endothelial apoptosis and cell migration [19–21]. In contrast, studies from several other groups show equivocal or even aggravating effects of exogenous E2 on TNF-mediated changes [19, 21–24]. Most of these studies also differ in the dose and duration of treatment with E2, 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 poorly understood [26–28]. Downstream of these receptors, TNF can activate various signaling and transcriptional pathways including nuclear factor (NF)-B and mitogenactivated protein (MAP) kinase (e.g. c-Jun N-terminal kinase 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 identified as a specific downstream target of TNFR2 [29]. NFB is a major pro-inflammatory transcription factor in the endothelium that is activated by various pro-inflammatory 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 2

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proteins known as inhibitory B (IB). Inflammatory events cause rapid phosphorylation and degradation of IB, leading to the release of p65/p50 and their subsequent 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 proinflammatory gene transcription [33]. Sp1 is another transcriptional factor commonly activated by E2, which is also a target of TNF action in the vasculature [34–36]. Targeting TNFRs and the downstream signaling pathways can affect the inflammatory process [37]. Yet little is known about the interactions between E2 and TNFactivated signaling pathways in the vascular endothelium. Given this background, we hypothesized that chronic E2 exposure would alter the endothelial inflammatory 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 inhibitor SP600125 (SP) and cyclodextrin-encapsulated 17--E2 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 laboratory 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. Guilbert’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 investigation 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.

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NS

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Fig. 1. E2 effects on TNF-induced endothelial ICAM-1 expression. a HUVEC mono-

layers were treated with/without E2 (10 nM) for 24 h prior to 4-hour stimulation with TNF (5 ng/ml). b, c HUVECs without (b) or with (c) E2 (10 nM) pre-treatment were washed and treated with NF-B inhibitor BAY (5  M), Sp1 inhibitor MitA (10 nM) 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 -tubulin (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).

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All subjects provided written informed consent before inclusion in the study. The umbilical vein was first flushed with PBS to remove blood clots and then HUVECs were isolated using a type 1 collagenase-containing buffer. The cells were grown in a humidified atmosphere at 37 ° C with 5% CO2/95% 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 nature of these cells was confirmed by staining for the endotheliumspecific marker, von Willebrand’s factor (data not shown). Our laboratory has published extensively using HUVECs as a model for the vascular endothelium [40–44].

E2, TNFR2 and Endothelial Inflammation

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Once the HUVEC monolayers were 80–90% confluent, we incubated these in phenol red-free M199 medium (to prevent estrogenic actions of phenol red) with 1% FBS for 24 h with/without E2 (10 nM). Following E2 treatment, the cells were washed once and stimulated with TNF (5 ng/ml). Western Blotting Western blotting was performed on the HUVEC lysates as described previously [44]. Bands for IB  and IB (rabbit polyclonal antibodies 9242 and 9248, respectively; Cell Signaling, Beverly, Mass., USA), VCAM-1 (rabbit polyclonal antibody sc8304; Santa Cruz Biotechnologies, Santa Cruz, Calif., USA), ICAM-1 (mouse monoclonal antibody sc-8439; Santa Cruz Biotechnologies), 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 Signaling) 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 antibodies 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 E2, no TNF) were loaded on every gel and the data were expressed as fold increase over the corresponding untreated control (i.e. no E2, no TNF). Immunofluorescence HUVECs were fixed in 3% formalin and immunostained using 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 associated with the cell membrane. Cells were visualized under an Olympus IX81 fluorescent microscope (Carson Scientific Imaging 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 E2, no TNF). One-way analysis of variance (ANOVA) with an appropriate 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 E2 and TNF), two-way ANOVA was used. A repeated-measure test was used

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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

E2 Alters the Transcriptional Pathways Mediating TNF-Induced ICAM-1 Expression We first examined the effects of 24-hour pre-treatment with E2 (10 nM) on TNF-induced ICAM-1 expression. We found that a 4-hour TNF stimulation significantly increased ICAM-1 protein levels (3- to 5-fold compared to the untreated control) in HUVECs, with no differences observed between the E2-treated and -untreated cells (fig. 1a). E2 alone had no effect on ICAM-1 levels. We also investigated the role of various transcriptional 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 nM) or JNK/AP-1 inhibitor SP (25 M) that were all used for 30 min prior to a 4-hour stimulation 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 E2) cells, ICAM-1 upregulation was almost entirely dependent on NF-B as this was completely (⬃97%) blocked in the presence of BAY (fig. 1b), similar to previously published reports [39, 48]. Surprisingly, in the E2-treated cells, NF-B only appeared to play a partial (⬃64%) role, while inhibition of JNK (and hence, the AP-1 transcriptional pathway) by SP significantly (⬃91%) attenuated ICAM-1 protein levels (fig. 1c). Results with cyclodextrin (the vehicle for E2) alone were not different from those in E2-free cells (online suppl. fig. S1, for all suppl. material, 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 E2-induced modulations 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. E2 Does Not Affect the Transcriptional Pathways Mediating TNF-Induced VCAM-1 Expression Next, we investigated the effects of 24-hour E2 pretreatment on TNF-induced VCAM-1 protein levels. We found that E2 treatment had no effects at all on TNF-mediated VCAM-1 expression (5- to 7-fold compared to conChakrabarti/Davidge

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Fig. 2. E2 effects on TNF-induced endothelial VCAM-1 expression. a HUVEC monolayers were treated with/without E2 (10 nM) for 24 h prior to 4-hour stimulation with TNF (5 ng/ml). b, c HUVECs without (b) or with (c) E2 (10 nM) pre-treatment were washed and treated with NF-B inhibitor BAY (5  M), Sp1 inhibitor MitA (10 nM) 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).

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trol; fig. 2a), which remained almost wholly dependent on NF-B signaling, both in the presence (⬃85%) or absence (⬃96%) of E2 (fig. 2b, c). These results suggested a highly specialized modulation of the pro-inflammatory signaling machinery by E2, such that upregulation of VCAM-1 remained unchanged while regulation of ICAM-1 was altered.

E2 Pre-Treatment Modulates TNF-Induced Degradation of IB without Affecting the JNK Pathway Given the altered roles of NF-B and JNK/AP-1 signaling pathways in the E2-treated cells, we then proceeded to examine the TNF-mediated activation profiles of these pathways in the presence/absence of prior E2 administra-

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Fig. 3. E2 effects on TNF-mediated NF-B activation patterns. HUVEC monolayers were pre-treated with/without E2 (10 nM, 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 IB  (a, c), IB (b, d) and -tubulin (loading control

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for all panels). Means 8 SEM from 4–5 independent experiments. * p ! 0.05, ** p ! 0.01 and *** p ! 0.001 vs. untreated control. IB  and IB levels were also analyzed by two-way ANOVA (e, f) to examine specific effects of E2 pre-treatment, TNF stimulation and the interactions (if any) between these.

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tion. NF-B activation is generally measured by the rapid degradation of intracellular IB on the administration of a suitable stimulus [49, 50]. There are two main forms of IB in the endothelium – namely IB and IB. IB is the form that is rapidly degraded by TNF, while the specific role for IB is less clear [51, 52]. We found that IB was almost completely (90–91% decrease) degraded within 15 min of TNF treatment in both E2-treated and -untreated cells. Even after 60 min, IB levels were only partially (38–54%) replenished, in agreement with previous work from our group [53]. In contrast, IB remained unaffected by TNF stimulation in the untreated cells, while it was partially degraded (42–48% decrease) on prolonged TNF treatment (45 and 60 min) only in the E2treated cells. Two-way ANOVA showed a significant effect of E2 on IB responses to TNF. These data are presented in figure 3 and indicate a key regulatory role for chronic E2 treatment on NF-B activation. We also examined the effects of E2 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 patterns were identical in E2-treated and -untreated cells (fig. 4a, b, e). In addition, no effects of E2 pre-treatment were observed on TNF-induced phosphorylation of cJun, a downstream target of JNK (fig. 4c, d, f). Interestingly, while the maximal response for JNK phosphorylation 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 activation. Collectively, these results suggest that the E2 effects on TNF-treated HUVECs were not due to altered JNK activity but rather involved altered NF-B signaling. E2 and TNF Differentially Regulate Expression Profiles of TNFR1 and TNFR2 We next studied the effects of E2 on TNFR profiles. Pro-inflammatory effects of TNF are generally mediated through TNFR1, while specific roles for TNFR2 are less clear [27]. Interestingly, neither E2 pre-treatment nor 4-hour stimulation with TNF caused any statistically significant changes in protein levels of TNFR1 (fig. 5a). In contrast, TNFR2 levels were increased by E2 treatment (⬃87% increase in Western blot), while two-way ANOVA showed significant interaction between E2 and TNF on TNFR2 expression (fig. 5b). Similarly, E2 pre-treatment (24 h) significantly increased (⬃60%) TNFR2 levels on the endothelial cell surface, as detected by immunofluorescence with an anti-TNFR2 antibody (fig. 5c), further E2, TNFR2 and Endothelial Inflammation

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supporting the Western blot findings. These results suggest a potential role for TNFR2 in mediating E2-induced modulation of TNF signaling in the endothelium. TNFR1 but Not TNFR2 Mediates Endothelial ICAM-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 E2-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. 6a). 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 rapidly phosphorylate Etk, suggesting that it was indeed acting through this receptor (fig. 6b). These findings indicate that endothelial ICAM-1 expression is largely dependent on TNFR1. While TNFR2 could potentially regulate the underlying 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 E2 treatment, where the total ICAM-1 levels remained unchanged (fig.  1a) while the transcriptional regulation was shifted from NF-B to JNK/AP-1. TNFR2 Is Critical for Alterations in TNF Signaling in the E2-Treated Endothelium Finally, we examined if TNFR2 signaling was indeed involved in mediating E2-specific effects on TNF-stimulated endothelium, namely the degradation of IB and JNK-dependent ICAM-1 expression. On E2-treated cells, TNFR1A alone did not significantly alter IB levels, while a combination of both TNFR1A and TNFR2A induced partial (35–46% decrease) IB degradation (fig. 7a), demonstrating the role of TNFR2 on this effect. In a similar vein, TNFR1A-mediated ICAM-1 expression (on E2-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. 7b). Despite its modulatory roles on TNFR1 effects, activation of TNFR2 alone could neither degrade IB (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 E2-mediated changes in endothelial signaling both on NF-B activity and ICAM1 expression. J Vasc Res

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monolayers were pre-treated with/without E2 (10 nM, 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 phospho-JNK (p-JNK) and total JNK (a, b) or phospho-c-Jun (p-c-Jun)

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and total c-Jun (c, d). Means 8 SEM from 5 independent experiments. * 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 E2 pre-treatment, TNF stimulation and the interaction (if any) between these (e, f).

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Fig. 5. Effects of E2 and TNF on endothelial TNF receptor expression. a, b HUVEC monolayers were treated with/without E2 (10 nM) 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 independent experiments. Data were also analyzed by two-way ANOVA to examine specific effects of E2 pre-treatment, TNF stimulation and the interactions (if any) between these. c HUVECs were treated with/without E2 (10 nM) for 24 h prior to fixation and immunostaining for TNFR2. Means 8 SEM from 3 independent experiments. * p ! 0.05 vs. untreated cells.

Discussion

Here we have shown the modulatory effects of chronic E2 administration on TNF-mediated ICAM-1 upregulation in the endothelium. E2 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 regulation of ICAM-1 was accompanied by de novo degradation E2, TNFR2 and Endothelial Inflammation

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of IB on TNF stimulation of the E2-treated cells. This work shows how E2 regulates inflammatory processes whereby the underlying signaling mechanisms are altered. TNF is a key pro-inflammatory cytokine involved in the pathogenesis of atherosclerosis and many other inflammatory 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, J Vasc Res

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Fig. 6. Selective activation of TNF receptors on endothelial ICAM1 expression. a HUVEC monolayers were pre-treated with/with-

out E2 (10 nM, 24 h) before stimulation with TNFR-specific agonists (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

51, 55]. The intricacies of TNF signaling, the roles of specific TNFRs and their regulation of NF-B are still incompletely understood. For example, while upregulation of both ICAM-1 and VCAM-1 in TNF-treated endothelium 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 inhibitor with some estrogen-like activity, reduced VCAM1 but not ICAM-1 expression in TNF-stimulated endothelial cells. The same study reported that sodium orthovanadate, a protein phosphatase inhibitor, also reduced TNF-induced VCAM-1 expression while it further aggravated upregulation of ICAM-1. Similarly, genipin (a modulator of peroxisome proliferator-activated 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-inflam10

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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 independent experiments. * p ! 0.05 and ** p ! 0.01, vs. untreated control.

matory cytokine IL-10 remain unaltered [58]. Collectively, these findings suggest that protein expression following 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 inhibitory proteins IB and IB. IB is known to be the main regulator of pro-inflammatory activity in response to diverse stimuli such as lipopolysaccharides, TNF and interferons. The regulatory effects of IB are less clear and variable [reviewed in ref. 32]. While most pro-inflammatory stimuli can induce degradation of IB, degradation of IB can occur only in a few instances [59–61]. Depending on the specific stimulus and the cell type, IB has been shown to be either synergistic with IB or acting quite differently [61, 62]. IB degradation occurs early in the inflammatory process, while that of IB is often delayed, a fact which may represent a more persistent activation of NF-B signaling in certain instances [63–65]. A study by Rao et al. [66] has recently shown that IB acts on a different set of genes Chakrabarti/Davidge

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6 p < 0.01

1.0

ICAM-1/-tubulin (fold change)

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* **

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2

0 in 0

– SP TNFR1A

Bo

th

:6

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in m

in Bo

th

:6

:4

0

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E2

TN

FR

1A FR TN

1A

:4

5

m

in

e on Al

Un

t re

at

ed

0

E2

IB -Tubulin a

Fig. 7. TNFR2 mediates E2 effects in TNF-treated endothelium. a HUVEC monolayers were pre-treated with E2 (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 IB and -tubulin (loading control). Means 8 SEM from 5–7 independent experiments. * p ! 0.05 and ** p ! 0.01 vs. E2 alone. b HUVEC monolayers were pre-treated

ICAM-1

-Tubulin b

with E2 (10 nM) for 24 h prior to 30-min treatment with/without JNK/AP-1 inhibitor SP (25  M) followed by stimulation with TNFR-specific agonists (TNFR1A and TNFR2A, 10 ng/ml each) for 4 h. Cell lysates were analyzed by Western blotting for ICAM-1 and -tubulin (loading control). Means 8 SEM from 5 independent experiments.

than IB, in addition to opposite effects on some of the same genes. Indeed, mice deficient in IB appear to be resistant to lipopolysaccharide-induced septic shock, suggesting a potential anti-inflammatory role for IB. Given this background, it is interesting to note that E2 pre-treatment caused TNF-induced IB 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 target c-Jun) remained unchanged in both E2-treated and -untreated 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 E2), JNK is also activated but does not regulate ICAM-1. On the other

hand, when NF-B activity is partially compromised (potentially due to IB 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 E2 pre-treatment. TNF signaling is involved in inflammation, oxidative stress and cell survival/apoptosis in the vascular endothelium. TNF responses are essential for wound healing and anti-bacterial defenses, while excessive and/or dysregulated responses can contribute to inflammatory diseases [13]. TNF exerts its effects through two different receptors: TNFR1 and TNFR2. Traditionally, most of the pro-inflammatory and pro-apoptotic TNF effects are believed to be dependent on TNFR1 [26]; not surprisingly, we also found that TNFR1 was responsible for the upregulation of the adhesion molecules in our system. However, TNFR2 levels were increased in the E2-treated cells,

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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 intracellular 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 IB kinases leading to the phosphorylation and subsequent degradation of IB molecules required for NF-B activity. The signaling complex soon dissociates from TNFR1 and then proceeds to activate a number of other pro-inflammatory signaling cascades, including JNK phosphorylation that leads to the activation of the AP-1 pathway [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 E2-treated cells, which allows for TNF-mediated IB 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 signaling pathways nor could it lead to ICAM-1 expression

[26]) yet it profoundly affected these processes when acting in concert with TNFR1 signaling. The actual signaling proteins/complexes that are involved in mediating the TNFR2 effects remain to be determined. Finally, it is not clear if the E2-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 TNFR2associated signaling complexes rather than a simple increase in TNFR2 levels. Future research would be directed to address these intriguing questions raised by our findings on E2-mediated alterations in TNFR2 signaling. In conclusion, E2 pre-treatment is shown to be a regulator of TNF effects in the vascular endothelium, which can help in our understanding of the sex-based differences 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 Heritage Foundation for Medical Research Scientist.

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