The Unfolded Protein Response Is an Important Regulator of Inflammatory Genes in Endothelial Cells
Objective— Oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine (oxPAPC) accumulates in atherosclerotic lesions and in vitro studies suggest that it mediates chronic inflammatory response in endothelial cells (ECs). The goal of our studies was to identify pathways mediating the induction of inflammatory genes by oxPAPC.
Methods and Results— Using expression arrays, quantitative polymerase chain reaction (PCR), and immunoblotting we demonstrate that oxPAPC leads to endoplasmic reticulum stress and activation of the unfolded protein response (UPR) in human aortic ECs. Immunohistochemistry analysis of human atherosclerotic lesions indicated that UPR is induced in areas containing oxidized phospholipids. Using the UPR inducing agent tunicamycin and selective siRNA targeting of the ATF4 and XBP1 branches of the UPR, we demonstrate that these transcription factors are essential mediators of IL8, IL6, and MCP1 expression in human aortic ECs required for maximal inflammatory gene expression in the basal state and after oxPAPC treatment. We also identify a novel oxPAPC-induced chemokine, the CXC motif ligand 3 (CXCL3), and show that its expression requires XBP1.
Conclusions— These data suggest that the UPR pathway is a general mediator of vascular inflammation and EC dysfunction in atherosclerosis, and, likely, other inflammatory disorders.
The development of atherosclerosis is associated with the production of pro-inflammatory cytokines and chemokines by endothelial cells (ECs), leading to monocyte recruitment and accumulation in the subendothelial space of arteries.1 Minimally oxidized low-density lipoprotein (MM-LDL), and its primary bioactive component, oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine (oxPAPC), have the ability to induce such inflammatory responses.2 Furthermore, oxPAPC accumulates in atherosclerotic lesions and other sites of chronic inflammation.3 The significance of oxPAPC is underscored by recent studies describing a significant association of oxidized phospholipids and the degree of coronary atherosclerosis in human population.4
The inflammatory effects of MM-LDL and OxPAPC are attributed in large part to the induction of chemokines such as IL8, MCP1 and CXC motif ligand (CXCL) chemokines,5–7 crucial for recruitment and migration of monocytes to the subendothelial space. MCP1, a member of the CC family of chemokines, has been shown to play a crucial role in monocyte transmigration,8 whereas IL8 and CXCL chemokines are known to mediate monocyte arrest to vascular endothelium.9,10 Additional studies have shown that oxPAPC induces the chemokine IL6, another important inflammatory regulator.11
The mechanisms of oxPAPC-mediated EC activation are complex and distinct from those induced by other inflammatory stimuli, such as bacterial lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha. For example, LPS activates the nuclear factor kappa B (NFκB) pathway, leading to a rapid induction of chemokines and adhesion molecules.12 Induction of IL8 by oxPAPC, however, is independent of NFκB pathway and, in contrast to LPS, results in a much more prolonged upregulation of IL8 expression.5 Previous studies have identified the c-src/STAT3 pathway as a mediator of IL8 induction by oxPAPC.13 Recently, it has been shown that oxPAPC, but not LPS, causes activation of the sterol regulatory element-binding protein (SREBP) and endothelial nitric oxide synthase (eNOS), both of which mediate induction of IL8.14,15 These pathways do not, however, fully explain the induction of IL8 expression by oxPAPC. The regulation of MCP1 expression and other oxPAPC-induced chemokines has been less studied and appears to differ from IL8.16 Because all of these chemokines are likely to participate in the unique monocyte-specific endothelial interactions, we sought to gain a more comprehensive understanding of the mechanisms by which OxPAPC regulates inflammation using microarray analyses.
Our present studies demonstrate that oxPAPC treatment causes endoplasmic reticulum (ER) stress, which results in activation of the unfolded protein response (UPR) in human aortic ECs. The UPR pathway is activated in cells under stress conditions that compromise the processing and folding of the proteins in the ER.17 Induction of the UPR has been recently demonstrated in atherosclerotic lesions of apolipoprotein E knockout mice and in macrophages that accumulate excessive amounts of cholesterol, a hallmark of atherosclerosis.18,19
We show, using selective siRNA-mediated targeting, that the UPR transcription factors ATF4 and XBP1 are essential mediators of several inflammatory genes implicated in atherosclerosis, including IL8, MCP1, IL6, and CXCL3. Our findings directly link the UPR and endothelial inflammation, and thus further highlight the potential importance of this adaptive stress response in the pathophysiology of atherosclerosis.
Materials and Methods
For detailed methods, please see the online Materials and Methods, available at http://atvb.ahajournals.org
Reagents and Antibodies
For oxPAPC and antibody sources, please see supplementary online Materials and Methods (available online at http://atvb.ahajournals.org). The EO6 antibody was a kind gift from Dr Joseph Witztum (UCSD). SiRNA duplexes were from Qiagen and PCR primers from Invitrogen (supplemental Table II).
Cell Culture, Treatments, Transfections, and siRNA Knock-Down Experiments
HAECs were isolated from aortic samples retrieved at the time of organ harvest for cardiac transplantation and used at passages 4 to 7. For siRNA experiments, cells were transfected with 5 nM siRNA using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s protocol.
Nuclear Protein Extraction and Immunoblotting
Nuclear protein extracts and immunoblotting were performed using standard procedures. When detecting ATF6 protein, proteosomal inhibitors were added to media during the 4-hour treatment.
Enzyme-Linked Immunosorbent Assay
IL8 levels in HAEC supernatants were measured using an enzyme-linked immunosorbent assay kit (Quantikine Immunoassay R&D Systems) as described.5
Expression Array Analysis
HAECs (triplicate) were cultured for 4 hours in media containing 0, 10, 30, or 50 μg/mL of OxPAPC and RNA expression profiles analyzed using Affymetrix U133A arrays. To select differentially expressed genes, we used cutoff criteria with P<0.05 (t test with Benjamini and Hochberg correction for the false discovery rate), absolute value fold change ≥1.5 and detection cell called present in at least one of the compared groups (control versus oxPAPC;50 μg/mL).
Formalin-fixed, paraffin-embedded tissue sections of coronary arteries from explanted hearts were blocked and stained overnight with ATF3, ATF4 antibody (1:50), or EO6 antibody (1:100), followed by secondary antibody and color-developed using DAB kit (Vector Laboratories).
Real-Time Quantitative PCR Analysis
Quantitative RT-PCR was performed using an ABI Prizm 7700 (Applied Biosystems) and SYBR Green detection (Sigma). Sequences of primers can be found in supplemental Table II.
Mapping the Genes Regulated by oxPAPC
We performed initial experiments by examining in detail the response of human aortic ECs (HAECs) after treatment with oxPAPC. Cultured primary HAECs were treated in triplicate with oxPAPC at concentrations of 0, 10, 30, or 50 μg/mL for 4 hours and analyzed for gene expression changes using Affymetrix expression arrays. We identified >700 genes differentially regulated by oxPAPC (50 μg/mL). Some of these genes, including inflammatory genes, such as IL8, IL6, MCP1, and CXCL1 (also known as GRO1) chemokines, have previously been shown to be regulated by oxPAPC, but the majority represented novel oxPAPC targets (Figure 1). Consistent with our previous studies, we observed that a significant number of genes regulated by oxPAPC were SREBP targets.14 These included LDLR, SQLE, INSIG1, HMGCS1, FDFT1, IDI1, and CYP51 (supplemental Table I). Interestingly, among the newly identified oxPAPC-regulated genes was CXCL3 (also known as GRO3), a chemokine from the same CXC family as IL8.
The UPR Pathway Is Induced by oxPAPC
The expression array analyses revealed that oxPAPC treatment induces a number of genes known to be regulated by the UPR pathway. These included 2 key mediators of the UPR signaling, ATF4 and XBP1, as well as several other known UPR target genes, including ATF3, C/EBPB, DDIT3 (Chop), HERPUD1, PPP1R15A, and the heat shock protein (Hsp) 40 family chaperones DNAJA1, DNAJB1,and DNAJB9 (Figure 1 and supplemental Table I).20–22 The induction of ATF4 and XBP1, together with upregulation of their target genes, was a strong indication that oxPAPC induces the UPR pathway in HAECs.
To confirm the expression array results, we incubated HAECs with oxPAPC and measured changes in mRNA levels of selected UPR genes by quantitative polymerase chain reaction (Q-PCR). OxPAPC treatment induced the mRNA levels of ATF4, ATF3, and XBP1 genes in a time-dependent manner (Figure 2). In unstressed cells, the degree of ATF4 mRNA translation is low, and this rapidly changes during UPR activation as a result of phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α).21 We observed increased phosphorylation of eIF2α within the first hour of oxPAPC treatment accompanied by induction in ATF4 protein levels (6-fold at 1 hour, 8-fold at 2 hours, and 5-fold at 4 hours) (Figure 2a and 2c). Not only are the XBP1 transcript levels induced but also the mRNA undergoes regulated splicing, resulting in formation of an active and highly stable transcription factor. Using specific primers,23 we selectively measured the level of the spliced XBP1 mRNA (sXBP1), which increased with time after exposure to oxPAPC (Figure 2e). In addition to ATF4 and XBP1, the activation of ATF6 constitutes the third branch mediating the UPR signaling.17 ATF6 is an ER-localized 90kD transmembrane protein, which undergoes regulated proteolytic cleavage under conditions of ER stress, resulting in liberation of an active 50-kD transcription factor. OxPAPC treatment of HAECs resulted in proteolytic processing of ATF6 as determined by reduced expression of the cytosolic 90-kD protein and increased expression of the nuclear 50-kD form (Figure 2d).
OxPAPC is composed of several active phospholipids, with 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (PEIPC) being the most active in terms of its ability to induce inflammation, as well as IL8 and HMOX1 expression (active at concentrations as low as 200 ng/mL).13,24 To determine whether PEIPC also activates the UPR, HAECs were treated for 4 hours with PEIPC (200 ng/mL) and the expression of the UPR genes was measured using Q-PCR. The PEIPC, but not unoxidized PAPC, treatment led to a significant induction of ATF3 (>150-fold), ATF4 (>2-fold), XBP1 (4-fold) (supplemental Figure I).
Immunolocalization of UPR Target Genes and Oxidized Phospholipids in Human Atherosclerotic Lesion
OxPAPC is known to accumulate in atherosclerotic lesions at concentrations 10-times higher (≈400 μg/g tissue) than those needed to activate UPR in vitro in human ECs.25 ATF3 and ATF4 were highly induced UPR targets by oxPAPC (Figure 2) and we used immunohistochemistry to examine their expression in human atherosclerotic lesions. Strong positive staining was found in endothelial cells in the inflammatory areas of the lesion shoulder but not in the fibrous cap area of the lesion (Figure 3a to 3c). Positive ATF3 and ATF4 staining was also seen in lesion areas containing foam cells.
To localize oxidized phospholipids with respect to ATF3 and ATF4 in lesions, we used the well-characterized autoantibody EO6, which recognizes epitopes on oxPAPC created by oxidizing PAPC at the sn2 position of the phospholipid backbone.26 The EO6 antibody heavily stained inflamed areas of the atherosclerotic plaque enriched in lipid deposits and foam cells (Figure 3d and 3e). Whereas there was little staining in endothelial cells, several areas in close proximity to the endothelial layer stained positively (supplemental Figure II). As judged from the staining pattern of the adjacent serial sections, there was a significant overlap in distribution and localization of EO6 and UPR markers. We observed no EO6 staining of the fibrous cap areas of the same sections, demonstrating that oxidized phospholipids are not present in this region (Figure 3f). These findings suggest that the UPR is induced in human atherosclerotic lesions, in the regions of the vessel containing oxidized phospholipids, consistent with our studies of isolated HAECs.
UPR Transcription Factors ATF4 and XBP1 Are Mediators of Inflammatory Gene Expression in HAECs
To determine whether the UPR contributes to the inflammatory gene expression in HAECs, we used an siRNA approach and selectively disrupted 2 key UPR mediators activated by oxPAPC, ATF4, and XBP1. HAECs lacking either of the 2 transcription factors were then treated with oxPAPC or a well-known UPR activator, tunicamycin,23 and analyzed for changes in expression of inflammatory genes induced by oxPAPC, IL8, CXCL3, IL6, and MCP1.
Oligo-based siRNA targeting of ATF4 effectively reduced endogenous ATF4 mRNA in untreated (71%), oxPAPC-treated (81%), and tunicamycin-treated (85%) cells (Figure 4a). The levels of ATF4 protein were also significantly reduced (Figure 4a). ATF4 silencing resulted in a significant decrease in IL8 mRNA levels in all treatment conditions (control 64%, oxPAPC 48%, tunicamycin 75%) (Figure 4b). The decrease in IL8 mRNA resulted in a corresponding reduction in the amount of secreted IL8 protein (data not shown). ATF4 inhibition had a similar inhibitory effect on IL6 and MCP1 expression (Figure 4d and 4e). CXCL3 expression was significantly reduced only in tunicamycin-treated cells (Figure 4c). With the exception of MCP1, tunicamycin treatment alone was sufficient to significantly induce mRNA expression of the studied inflammatory genes, but not to a degree seen with the oxPAPC treatment. Interestingly, although ATF4 siRNA had a significant impact on the absolute level of IL8, IL6, and MCP1 expression in oxPAPC-treated cells, the degree of inducibility of these genes by oxPAPC was not affected.
These effects were then compared with HAECs transfected with XBP1 siRNA. OxPAPC and tunicamycin treatment increased the ratio of spliced to unspliced (uXBP1) form of XBP1 protein, as expected. The siRNA targeting of XBP1 resulted in a significant decrease in total and spliced mRNA (≈90%), as well as the protein levels (Figure 5a; supplemental Figure III). The disruption of XBP1 had an even more striking effect, resulting in significant inhibition of IL8, IL6, CXCL3, and MCP1 expression in untreated, oxPAPC-treated or tunicamycin-treated cells (Figure 5b to 5e). Importantly, in cells treated with oxPAPC, the disruption of XBP1 resulted in downregulation of the studied inflammatory genes to the degree comparable to basal levels (untreated cells expressing XBP1). Similar to ATF4, however, the XBP1 silencing did not affect the fold induction in response to oxPAPC. The siRNA targeting was not only potent but also selective because we observed no significant downregulation of other genes regulated by oxPAPC, such as LDLR and HMOX1 (supplemental Figure III).
We present evidence that exposure of HAECs to oxidized phospholipids results in the induction of UPR and that 2 key UPR mediators, ATF4 and XBP1, directly participate in modulating inflammatory responses important in atherosclerosis.
UPR signaling is vital for normal maintenance of the ER homeostasis of cells as well as their response to stresses that negatively affect ER function. Interestingly, previous studies have shown that exposure of cells to oxidants can lead to ER stress, and induction of UPR serves a protective role against oxidative stress.27 Furthermore, UPR induction has also been demonstrated in macrophages accumulating excessive amounts of cholesterol and in endothelial cells treated with peroxynitrite, both likely to be important factors in atherosclerotic disease development.19,28
The transcription factors ATF4, XBP1, and ATF6, are key mediators of UPR signaling.17 Studies in cells lacking ATF4 have implicated it in regulation of amino acid metabolism genes, glutathione biosynthesis, and resistance to oxidative stress.27 XBP1 and ATF6, however, control regulation of genes required for protein folding, maturation, and degradation in the ER.20,29 OxPAPC treatment increased ATF4 protein levels, induced XBP1 splicing, and resulted in cleavage of ATF6 in HAECs, indicating that all 3 branches of the UPR were activated within the span of 4-hour treatment.
Prolonged ER stress can lead to cell toxicity and apoptosis. It is important to note that oxPAPC at concentrations up to 50 μg/mL did not affect cell viability.6 As judged by induction of ATF4 and XBP1, the activation of UPR by oxPAPC was significant, but considerably lower than that seen with tunicamycin (Figures 4 and 5⇑). Moreover, we observed no changes in caspase-3 activity, and the removal of oxPAPC after 4 hours resulted in normal cell division without measurable cell toxicity (unpublished observations). Consistent with this, we observed only very modest upregulation (2-fold) of the pro-apoptotic mediator of UPR, DDIT3 (Chop), which was highly induced by tunicamycin (30-fold) (supplemental Figure III).19 Phosphorylation of eIF2α represents the early stages of UPR activation, whereas induction of ER chaperone GRP78 the later stage.29 OxPAPC failed to induce GRP78 in HAECs within 4 hours of treatment, further indicating that the UPR is likely to correspond to the early, nonapoptotic stages. In addition, ATF4 or XBP1 gene silencing did not result in increased susceptibility of HAECs to apoptotic cell death or increased cell toxicity when treated with oxPAPC (data not shown).
The mechanisms by which oxidized phospholipids induce UPR are unclear. We considered the possibility that the UPR in HAECs could be activated by mechanisms involving oxPAPC-mediated cholesterol depletion, a scenario opposite to the UPR induction by cholesterol loading in macrophages.14,19 Our data, however, indicate that this is not the case, because in contrast to SREBP activation (as judged by decrease in LDLR and INSIG1 expression), the UPR induction by oxPAPC was not prevented by addition of excess cholesterol (supplemental Figure IV). Recent data indicate that prostaglandin (PG) E2 receptor subtype 2 (EP2) is involved in oxPAPC-mediated activation of HAECs to bind monocytes.30 However, in our preliminary studies, the specific EP2 agonist butaprost failed to induce UPR in HAECs, suggesting that UPR is activated by distinct mechanisms. OxPAPC leads to an increase in oxidative stress which may, at least in part, explain the UPR induction.15,31
In our genetic studies of HAECs derived from separate donors, we noticed significant correlations between inflammatory and UPR genes, including ATF4 and XBP1 (data not shown). Present studies in primary HAECs deficient in these 2 key UPR transcription factors led to an important finding, demonstrating that both ATF4 and XBP1 are necessary mediators of IL8, as well as IL6 and MCP1 expression. In contrast, expression of CXCL3, was affected primarily by XBP1. An unexpected finding of these studies was the observation that ATF4 and XBP1 appear to be important for basal production of the studied cytokines rather than their inducibility by oxPAPC. However, alterations in the level of expression of ATF4 and XBP1 are likely to be of importance in determining the overall maximal level of inflammatory response in many conditions, including OxPAPC-treated HAECs (Figures 4 and 5⇑). Thus, our data suggest that, at least in ECs, the mechanism controlling the basal ER homeostasis appears to be tightly linked to regulation of inflammatory gene transcription.
Interestingly, findings by Zhang et al indicate that UPR is activated by LPS in hepatocytes, leading to induction of inflammatory acute phase response genes, via activation of liver-specific transcription factor CREBH.32 LPS-mediated or oxPAPC-mediated inflammatory gene induction in HAECs is not likely to involve CREBH, because its expression is restricted to liver cells. In addition, LPS does not appear to activate UPR in HAECs while potently inducing inflammatory gene expression (unpublished observations).
The relevance of the activation of UPR in HAECs by oxPAPC is supported by our immunohistochemistry analysis of human atherosclerotic lesions. These studies indicated that ATF3 and ATF4 staining was restricted to inflammatory areas of human atherosclerotic lesions containing oxidized phospholipids, with high-positive staining of ECs and foam cells (Figure 3). High EO6 staining of foam cells in lesion areas could be explained by the fact that macrophages, but not ECs, routinely take up excessive amounts of cholesterol and oxidized lipids, a hallmark of atherosclerosis.26 Our findings are consistent with the hypothesis that UPR is induced by oxidized phospholipids in endothelial cells. However, besides the UPR, other stress factors have been shown to affect ATF3 and ATF4 expression, which could also contribute to their observed induction in atherosclerotic lesions.
Sources of Funding
This work has been supported by NIH grants HL30568 (A.J.L. and J.B.), HL64731 (J.B.), an unrestricted research award from Bristol-Myers Squibb (A.J.L.), an AHA Postdoctoral Fellowship award (P.S.G.), an AHA Predoctoral Fellowship award (N.M.G.), and the Laubisch Fund, UCLA.
Original received April 7, 2006; final version accepted July 18, 2006.
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