Apolipoprotein E Interrupts Interleukin-1β Signaling in Vascular Smooth Muscle Cells
Objectives— Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1β (IL-1β), a proinflammatory cytokine present in atherosclerotic lesions.
Methods and Results— IL-1β-induced expression and activation of inducible nitric oxide synthase and cyclooxygenase-2 were inhibited by apoE in vascular smooth muscle cells (VSMCs). These inhibitory effects were linked to the suppression of both NF-κB and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1β signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1β signaling intermediates revealed that NF-κB transactivation was inhibited by apoE in MyD88- and IRAK1- but not in TRAF6-transfected cells. Furthermore, apoE prevented IRAK1 phosphorylation and IRAK1-TRAF6 but not MyD88-IRAK1 complex formation. Inhibitory effects of apoE on IL-1β signaling were abolished after silencing LDL receptor-related protein-1 (LRP1) expression with siRNA. In addition, inhibitors of adenylyl cyclase and protein kinase A (PKA) restored IL-1β signaling in apoE-treated VSMCs, whereas apoE stimulated PKA activity. ApoE inhibited VSMC activation in response to IL-18 but not to tumor necrosis factor-α or polyinosinic:polycytidylic acid.
Conclusion— ApoE targets IRAK-1 activation and thereby interrupts IL-1β and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.
Apolipoprotein E (apoE) is a major protein component of plasma lipoproteins and plays an important role in preventing atherosclerosis. The antiatherogenic effects of apoE are usually attributed to its ability to promote cholesterol efflux from peripheral cells for reverse cholesterol transport and to facilitate hepatic clearance of very low density lipoprotein (VLDL) and chylomicron remnants. However, apoE protects against atherosclerosis even in experimental settings, in which its effects on plasma cholesterol are negligible. For instance, transgenic expression of apoE in arterial wall led to inhibition of atheroma formation without affecting plasma lipoprotein profile.1,2 Conversely, acceleration of atherosclerosis was observed in apoE+/+ animals after transplantation of bone marrow from apoE-deficient mouse.3 These results suggest that apoE directly inhibits the development of atherosclerosis in a manner independent of its role in cholesterol transport.
Atherosclerosis is commonly regarded as a chronic inflammatory disease. Inflammation mediators such as cytokines and chemokines were shown to significantly contribute to the formation of atherosclerotic lesions, whereas antiinflammatory factors play the opposite role. ApoE has been previously suggested to suppress the expression of adhesion molecules on endothelial surface and thereby to prevent monocyte recruitment into the arterial intima.4 However, little information is available concerning the involvement of apoE in initiation and perpetuation of inflammation within the vascular wall. In the present study, we examined the influence of apoE on cellular effects of interleukin-1β (IL-1β), a proinflammatory cytokine produced by macrophages and present in atherosclerotic lesions. Our results indicate that apoE interferes with intracellular signaling in response to IL-1β. Along this way, apoE prevents IL-1β-induced activation of vascular smooth muscle cells (VSMCs), constituting an important component of the inflammatory sequel in the vascular wall.
VSMCs derived from rat thoracic aortas were maintained in Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum. Human aortic smooth muscle cells (hASMCs, third passage) were purchased from a commercial supplier and cultured in SmGM-2 medium. In most experiments cells were preincubated for 1 hour with apoE (0 to 10 μg/mL) and stimulated with 10 ng/mL IL-1β.
Transient Transfections and Reporter Assays
Starved VSMCs were transfected with p(κB)5-Luc and p(AP-1)7-Luc plasmids using LipofectAmine 2000 (Invitrogen). Cotransfection experiments with MyD88, IRAK-1, or TRAF-6 expression plasmids were carried out by nucleofection. Lysates were assayed for luminescence intensity using Luciferase Assay System (Promega). β-galactosidase activity was used to normalize luciferase activity.
The siRNA vectors based on pGeneClip-hMGFP and expressing 4 individual predesigned siRNA plus GFP as a reporter gene were transfected into VSMCs with LipofectAmine 2000. The overall transfection efficiency was assessed by fluorescence microscopy.
Data are presented as means±SD from at least 3 separate experiments or as results representative for at least 3 repetitions, unless indicated otherwise. Comparisons between the groups were performed with 2-tailed Student t test. Probability values less than 0.05 were considered significant.
Detailed description of all methods can be found in the supplemental materials, available online at http://atvb.ahajournals.org.
ApoE Inhibits IL-1β-Induced iNOS and COX-2 Expression in VSMCs
Addition of IL-1β to VSMCs led to accumulation of iNOS and COX-2 mRNA, which was markedly reduced in cells pretreated with apoE (Figure 1A). This was accompanied by decreased iNOS and COX-2 synthesis. As shown in Figure 1B, the amounts of both enzymes were elevated after stimulation with IL-1β. ApoE alone failed to affect iNOS and COX-2 expression, but reduced the amounts of both enzymes, when added to VSMCs before IL-1β stimulation. The suppressing effects of apoE on IL-1β-induced iNOS and COX-2 synthesis were reflected by reduced enzymatic activities: IL-1β-stimulated nitrite/nitrate and prostaglandin E2 (PGE2) levels were reduced in a concentration-dependent fashion in the presence of apoE (Figure 1C).
The inhibitory effects exerted by apoE on iNOS and COX-2 were additionally assessed in hASMCs. Whereas IL-1β failed to induce iNOS expression and nitrite/nitrate generation in hASMCs, both COX-2 expression and PGE2 production were potently stimulated by IL-1β and these effects were completely abolished in the presence of apoE (Figure 1D; see also supplementary materials.). Endotoxin contamination did not account for the inhibitory effects of apoE on VSMCs activation (see supplementary materials).
ApoE Inhibits IL-1β-Induced NF-κB Activation and IκB Phosphorylation in VSMCs
As a first step to investigate mechanisms underlying the inhibitory effect of apoE on iNOS and COX-2 expression, we examined the activity of the transcription factor NF-κB, which controls iNOS and COX-2 expression in VSMCs. To this aim, the IL-1β-induced binding of p65/RelA, a component of the NF-κB complex, to NF-κB binding DNA sequence was investigated in VSMCs in the presence or absence of apoE. As shown in Figure 2A, IL-1β activated NF-κB DNA binding and this effect was inhibited in VSMCs pretreated with apoE. To verify the inhibitory effect of apoE on NF-κB activation, transient transfection assays were performed using NF-κB-responsive reporter vector. Treatment of VSMCs with IL-1β increased NF-κB transcriptional activity, whereas apoE alone was ineffective. However, the stimulatory effect of IL-1β was reversed by apoE in VSMCs and hASMCs (Figure 2B).
NF-κB activation is preceded by phosphorylation and degradation of IκB, a component of the NF-κB complex.5 Therefore, we investigated total and phosphorylated IκB in VSMCs exposed to IL-1β in the absence or presence of apoE. Consistent with previous studies, IL-1β reduced total amount and increased phosphorylated IκB in VSMCs (Figure 2C). Both effects were reduced in cells pretreated with apoE, and IκB phosphorylation was inhibited in a concentration-dependent manner (Figure 2C). Inhibitory effects of apoE were also seen in hASMCs (Figure 2C). IκB phosphorylation is mediated by IκB kinases (IKK) α and β. Figure 2D demonstrates that apoE inhibited IL-1β-increased phosphorylation of IKK-α.
ApoE Inhibits IL-1β-Induced AP-1 Activation in VSMCs
In addition to NF-κB, the expression of iNOS and COX-2 in VSMCs is transcriptionally controlled by activating protein-1 (AP-1). The IL-1β-induced activation of AP-1 and upstream regulatory kinases p38MAPK and JNK were inhibited in the presence of apoE (see supplementary materials).
ApoE Prevents IL-1β-Induced IRAK-1/TRAF-6 Complex Formation and Inhibits IRAK-1 Phosphorylation in VSMCs
The reduced activity of both NF-κB and AP-1 in the presence of apoE suggests that the interruption of IL-1β signaling occurs upstream of transcription factors. Therefore, we assessed the effects of apoE on the function of myeloid differentiation factor 88 (MyD88), IL receptor-associated kinase-1 (IRAK-1), and TNF-α receptor-associated factor-6 (TRAF-6)-signaling intermediates consecutively activated in VSMCs on stimulation with IL-1β. To this aim, VSMCs were cotransfected with plasmids encoding wild-type MyD88, IRAK-1, or TRAF-6 together with the p(κB)5-Luc reporter plasmid. Transfection of cells with either of 3 IL-1β signaling intermediates enhanced NF-κB transcriptional activity (Figure 3A). ApoE reduced NF-κB activity in VSMCs transfected with MyD88 or IRAK-1 but not with TRAF-6.
To further narrow the location of the signaling interference produced by apoE, we examined the formation of MyD88-IRAK-1 and IRAK-1-TRAF-6 complexes in response to IL-1β. We transfected an expression vector for Myc-IRAK-1 into VSMCs and assessed the anti-Myc immunoprecipitate for the presence of MyD88 or TRAF-6. As shown in Figure 3B, IRAK-1 coimmunoprecipitated MyD88 or TRAF-6 in VSMCs exposed to IL-1β. Preincubation of cells with apoE slightly increased MyD88-IRAK-1 complex formation but severely impaired the IRAK-1-TRAF-6 complex formation. Because activation of IRAK-1 precedes the formation of IRAK-1-TRAF-6 complex, we assessed the activation state of IRAK-1 in IL-1β-stimulated cells in the presence or absence of apoE exploiting the property of IRAK-1 to autophosphorylate. As shown in Figure 3C, immunoprecipitation of IRAK-1 from VSMCs exposed to IL-1β led to appearance of a typical migration pattern consisting of several bands corresponding to sequential steps of IRAK-1 phosphorylation at multiple sites.6 The IL-1β-induced IRAK-1 phosphorylation was inhibited by apoE.
The activation of IRAK-1 is negatively regulated by other members of the IRAK family as well as by the suppressor of cytokine signaling-1 (SOCS-1).7 ApoE failed to induce the expression of IRAK-2, IRAK-M, and SOCS-1 in VSMCs (Figure 3D).
LDL Receptor-Related Protein 1 and Protein Kinase A Are Involved in the Inhibitory Effects of ApoE on IL-1β-Induced Signaling in VSMCs
Next, signaling pathways utilized by apoE to interrupt IL-1β signaling were characterized. Initially, we compared apoE with apolipoprotein A-I (apoA-I), which interacts with some but not all apoE receptors. As shown in Figure 4A, the activation of NF-κB DNA binding and NF-κB transcriptional activity as well as the phosphorylation of IκB in response to IL-1β remained unchanged in the presence of apoA-I. To further analyze the receptor involvement in the inhibitory effects of apoE, we made either use of receptor associated protein (RAP), which interferes with apoE binding to the low density lipoprotein (LDL) receptor family members, or heparinase, which abolishes apoE binding to heparan sulfate-containing proteoglycans. As shown in Figure 4B, inhibitory effects of apoE on the activation of NF-κB DNA binding, NF-κB transcriptional activity, and IκB phosphorylation in response to IL-1β were reversed in the presence of RAP, but not heparinase. To address more specifically the identity of the receptor mediating inhibitory effects of apoE, we downregulated the expression of LDL receptor-related protein 1 (LRP1)—a member of LDL receptor family responsible for the suppressing effects of apoE on VSMCs proliferation8—with a combination of 4 vectors encoding distinct siRNAs specific for LRP1 and a green fluorescent protein (GFP) as a reporter gene. As shown in Figure 4C, VSMCs were effectively transfected with siRNA-encoding vectors and the expression of LRP1 was substantially reduced as assessed by polymerase chain reaction (PCR). Figure 4D demonstrates that the inhibitory effects of apoE on NF-κB transcriptional activity and IκB phosphorylation in response to IL-1β were absent in VSMCs transfected with LRP1-siRNA.
ApoE binding to LRP1 is associated with activation of protein kinases protein kinase A (PKA) and Akt.8,9 In addition, Akt and calcium/calmodulin-dependent protein kinase (CaMK) phosphorylate and inhibit IRAK-1.10 As shown in Figure 5A, the inhibitory effects of apoE on NF-κB and AP-1 activity as well as IκB and JNK phosphorylation in response to IL-1β were attenuated in VSMCs pretreated with H89, the pharmacological inhibitor of PKA, and SQ22536, the inhibitor of adenylate cyclase, which is an upstream activator of PKA. By contrast, KN93, the CaMK inhibitor, and LY294002, the Akt inhibitor, failed to interfere with the inhibitory effects of apoE. Figure 5B demonstrates that apoE induced activation of PKA comparable to that brought about by 8Br-cAMP—a cell-permeable PKA activator. The apoE-induced PKA activation was abolished by H89 and reduced by SQ22536. Moreover, the inhibitory effects of apoE on IRAK-1 phosphorylation and IRAK-1-TRAF-6 complex formation in response to IL-1β were emulated by pretreatment of VSMCs with 8Br-cAMP (Figure 5C and 5D).
Effect of ApoE on VSMC Activation in Response to IL-18, TNF-α, and Polyinosinic:Polycytidylic Acid
In addition to IL-1β, apoE inhibited VSMC activation induced by IL-18, which predominantly exploits MyD88-IRAK-1-TRAF-6 signaling pathway,11 but not by TNF-α and polyinosinic:polycytidylic acid (poly(I:C)) (Toll-like receptor 3 [TLR-3] agonist), which use IRAK-1 or TRAF-6 as auxiliary signaling molecules12,13 (see supplementary materials).
ApoE protects against atherosclerosis, and antiatherogenic effects of apoE are partly explained by the capacity of this apolipoprotein to interact with and to modulate VSMC function. For instance, apoE inhibits VSMC proliferation and migration and promotes VSMC-dependent extracellular matrix expansion.14–17 However, as yet no effects of apoE on the inflammatory activation of VSMCs have been reported. In the present study we show that the expression and activation of COX-2 and iNOS—2 enzymatic markers of inflammation—in response to IL-1β was downregulated in the presence of apoE. The inhibitory effects of apoE were seen both in rat and human VSMCs and occurred within a concentration range expected in the vasculature.18 Taken together, these findings demonstrate that apoE exerts suppressing effects on the inflammatory activation of VSMCs.
Previous studies highlighted the central role of NF-κB and AP-1 in the regulation of COX-2 and iNOS expression.5 These transcription factors are activated by inflammatory molecules commonly released in the vascular environment and detected within atherosclerotic lesions.19,20 Our results show that interaction of apoE with VSMCs leads to impaired NF-κB and AP-1 promoter binding and transcriptional activation. To our knowledge, this is the first demonstration of the antagonistic effect of apoE on NF-κB and AP-1 activation evoked by proinflammatory stimuli in vitro. Several molecular mechanisms can be invoked to explain the inhibitory influence of apoE on NF-κB and AP-1 activation. Decreased promoter binding or nuclear translocation of p65/RelA or c-jun or reduced bioavailability of coactivators such as CBP/p300 were all postulated to account for the negative regulatory effects exerted on NF-κB and AP-1 activation by other antiatherogenic factors.21–24 Whereas our results do not preclude that apoE perturbs 1 or more of the above mechanisms, the inhibition of IL-1β-induced IκB and c-jun phosphorylation as well as IKK-α and JNK activation suggests that apoE interferes with upstream signaling pathways controlling both NF-κB and AP-1. The activation of IL-1 receptor (IL1-R1) results in the recruitment of IRAK-1 to receptor complex via an adapter molecule MyD88, followed by the formation of IRAK-1-TRAF-6 complex, which in turn is involved in NF-κB and AP-1 activation.25 The present study demonstrates that apoE inhibits transcription factor activation in MyD88 and IRAK-1 but not in TRAF-6-overexpressing cells and that this apolipoprotein prevents IRAK-1-TRAF-6 but not IRAK-1-MyD88 complex formation. Taken together, these results suggest that apoE disrupts IL-1β signaling upstream to NF-κB and AP-1 and identifies IRAK-1 as a molecular target of antiinflammatory effects of apoE in VSMCs.
The mechanism underlying the inhibitory effects of apoE on IRAK-1 function remains to be elucidated. During activation IRAK-1 becomes phosphorylated by IRAK-4, which terminates its interaction with MyD88 and initiates the aggregation with TRAF-6.6,26,27 The formation of the IRAK-1-TRAF-6 complex is interrupted by IRAK-M, IRAK-2, or SOCS-1, and factors increasing their expression suppress NF-κB and AP-1 activation. However, in the present study expression of IRAK-M, SOCS-1, or IRAK-2 remained unaltered in VSMCs exposed to apoE. As apoE prevented IRAK-1 phosphorylation, it may be assumed that the inhibition of IRAK-1 function occurs before its dissociation from the IL-1-R1-MyD88 complex. Posttranslational modifications such as phosphorylation by Akt or CaMK were previously proposed to prevent IRAK-1 autophosphorylation.10 However, in the present study the apoE-induced suppression of IκB phosphorylation and NF-κB activation were both retained in the presence of Akt and CaMK inhibitors, thus excluding the involvement of both kinases in the inhibitory effects of apoE on IL-1β signaling. Hence, apoE seems to target IRAK-1 function by other mechanisms that may include functional modifications of either IRAK-1 or IRAK-4.
The preservation of apoE-induced NF-κB suppression in cells overexpressing MyD88 or IRAK-1 suggests that its inhibitory effects do not arise as a consequence of IL-1β displacement from IL-1 receptor. As apoE absorbs cholesterol from cell membranes, it might induce a perturbation of microenvironment, in which IL-1 receptor complex is localized. However, apoA-I—another apolipoprotein depleting membrane cholesterol—failed to affect IL-1β signaling, suggesting that the inhibitory effects are apoE-specific. It is more likely that the interaction of apoE with cells generates intracellular signals that interfere with IRAK-1 activation. Previous studies demonstrated that members of the LDL receptor family and heparin-sulfate proteoglycans serve as apoE binding partners in VSMCs.8,15–17 The observation that the suppressing effects of apoE on IL-1β-induced IκB phosphorylation and NF-κB binding and activation were abrogated in the presence of RAP or after silencing of LRP1 expression with specific siRNA but remained intact in heparinase-treated cells suggests that the interaction of apoE with LRP1 is required for the inhibition of IL-1β signaling. Previous studies demonstrated that the stimulatory effects of apoE on cAMP synthesis and PKA activation in VSMCs are mediated by LRP-1.8 Our present results extend these observations by showing that inhibitory effects of apoE on IL-1β-induced NF-κB and AP-1 activation are reversed after inhibition of adenylate cyclase or PKA.
The demonstration of the inhibitory influence of apoE on IL-1β-induced VSMC activation may have far-reaching consequences for the understanding its atheroprotective effects in humans and experimental animals. The permanent disinhibition of IL-1β signaling ought to be taken into consideration while interpreting results obtained with apoE-deficient mice. Moreover, IL-1β is a proinflammatory cytokine that promotes cholesterol accumulation by macrophages, expression of endothelial adhesion molecules, and VSMC migration at sites of vascular injury. Genetic ablation of IL-1β or IL-1R1 reduces lesion formation.28,29 Conversely, IL-1RA, a naturally occurring inhibitor of IL-1β signaling, decreases the severity of atherosclerosis.30 Hence, the inhibition of IL-1β signaling may contribute to the antiatherogenic potential of apoE. However, IRAK-1—the principal target of apoE antiinflammatory signaling in VSMCs—is shared by various signal transduction pathways used by proatherogenic factors. For instance, IL-18, a cytokine aggravating atherosclerosis in mice, promotes IRAK-1 phosphorylation and IRAK-1-TRAF-6 complex formation.11 Moreover, MyD88-IRAK-1-TRAF-6 pathway is also exploited, albeit to various extent, by TNFα and by Toll-like receptors (TLRs) that both initiate formation of atherosclerotic lesions.12,13,31,32 The present study demonstrates that IL-18 signaling is negatively regulated by apoE. By contrast, proinflammatory effects of TNFα and poly(I:C)—the TLR-3 ligand—were not influenced by apoE. The considerable diversity of TNFα and TLR signaling may account for this observation. Whereas MyD88-IRAK-1-TRAF-6 represents a primary signal transducing module in cells exposed to IL-1β and IL-18, various mutually redundant pathways are switched on upon stimulation with TNF-α or TLR ligands. For instance, TAB-1/2, TAK-1, and TICAM-1 may be preferentially used by TLR-3 instead of MyD88, IRAK-4, and IRAK-1 to secure TRAF-6 recruitment to the TLR receptor.13 Likewise, activation of JAK-STAT pathway rather than NF-κB is involved in TNF-α-induced VSMC activation.33
In conclusion, the present study demonstrates that by targeting IRAK-1, apoE restricts cytokine-induced activation of VSMCs. This antiinflammatory effect may significantly contribute to the antiatherogenic potential attributed to this apolipoprotein.
The expert technical assistance of Katrin Tkotz and Cornelia Elzenheimer-Richter is gratefully acknowledged.
Sources of Funding
This work was supported by a grant NO110441 from the Innovative Medizinische Forschung (IMF) to J.-R.N. and grant EC116/3-6 from the Deutsche Forschungsgemeinschaft (DFG) to A.v.E. and G.A. R.T was supported by a grant BMBF0313040C from German Ministry for Education and Science (BMBF).
Original received August 17, 2006; final version accepted May 3, 2007.
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