Published online before print January 25, 2007, doi: 10.1161/01.RES.0000258877.57836.d2
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© 2007 American Heart Association, Inc.
Integrative Physiology |
Absence of the G Protein–Coupled Receptor G2A in Mice Promotes Monocyte/Endothelial Interactions in Aorta
From the Robert M. Berne Cardiovascular Research Center (D.T.B., A.M.W., M.S., T.D., J.L., C.C.H.), Division of Cardiovascular Medicine (C.C.H.), and Department of Pharmacology (C.C.H.), University of Virginia, Charlottesville.
Correspondence to Catherine C. Hedrick, PhD, Cardiovascular Research Center, University of Virginia, PO Box 801394, 415 Lane Rd, MR5 Rm G123, Charlottesville, VA 22908. E-mail cch6n{at}virginia.edu
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Abstract |
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The G protein–coupled receptor G2A is highly expressed on macrophages and lymphocytes and has been localized to atherosclerotic plaques. We examined the role of G2A in modulating monocyte/endothelial interactions in the vessel wall. We measured adhesion of WEHI 78/24 monocytes to aortas of C57BL/6 (B6) and G2A-deficient (G2A–/–) mice using an ex vivo adhesion assay. G2A–/– mice had 10-fold elevations in adhesion of monocytes to aortas. Injection of GFP-expressing wild-type macrophages into B6 and G2A–/– mice in vivo showed increased macrophage accumulation in the aortic wall of G2A–/– mice. We isolated aortic endothelial cells (ECs) from B6 and G2A–/– mice and found a 2-fold increase in intercellular adhesion molecule-1 and E-selectin surface expression on G2A–/– ECs using flow cytometry. Using ELISA, we found a 3-fold increase in interleukin-6 and monocyte chemoattractant protein-1 production by G2A–/– ECs compared with B6 ECs. We found a dramatic increase in nuclear localization of the p65 subunit of nuclear factor
B in G2A–/– ECs. Transfection of G2A into G2A–/– ECs to restore normal expression levels reduced p65 nuclear localization to 35%. Restoration of G2A expression in G2A–/– ECs significantly reduced intercellular adhesion molecule-1 and endothelial selectin surface expression and reduced monocyte chemoattractant protein-1 and interleukin-6 production. Restoring G2A to G2A–/– ECs reduced monocyte adhesion by 80% compared with G2A–/– ECs in a flow chamber assay. Absence of G2A in endothelium results in proinflammatory signaling and increased monocyte/endothelial interactions in the aortic wall. Thus, endothelial G2A expression may aid in prevention of vascular inflammation and atherosclerosis.
Key Words: G protein–coupled receptor • NFB • endothelium • ICAM-1
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Introduction |
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A key early event in atherosclerosis is the increased interaction of monocytes with endothelial cells in the vessel wall.1 Monocytes are the primary inflammatory cells localized to human atherosclerotic plaques and play a major role in atherosclerotic plaque progression.2 Monocytes roll along the vascular endothelium and are activated by endothelial-derived soluble and/or surface-bound chemokines, resulting in enhanced affinity of LFA-1 (leukocyte function–associated molecule-1) and very late antigen (VLA-4) (via
4ß1) integrins for their endothelial receptors, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, respectively. Interaction of these monocytic integrins with ICAM-1 and VCAM-1 on the endothelial cell surface causes the monocyte to tether and firmly adhere to the endothelium, where it can subsequently transmigrate into the subendothelial space.3,4 The G protein–coupled receptor G2A was originally identified as a stress-inducible receptor that was induced in pre-B cells by the Bcr-Abl oncogene.5 G2A overexpression in fibroblasts causes cell cycle arrest at the G2 phase of mitosis (thus the name G2A for G2 Accumulation).5 G2A expression has been shown to attenuate Bcr-Abl oncogene–mediated cell proliferation, whereas mice lacking G2A have increased susceptibility to oncogene-induced leukemia.6 G2A was previously reported to be a specific receptor for lysophosphatidylcholine (LPC),7 but this report was later retracted.8 Overexpression studies suggest that G2A is redistributed in the cell in response to LPC treatment.9 However, Shimizu and colleagues suggest that LPC antagonizes G2A action.10 Recently, evidence suggests a role for G2A as a proton sensor in the cell.11,12 G2A, as well as other receptors within the OGR1 family (TDAG8, GPR4, and OGR1), can respond to changes in extracellular pH,12 although G2A is less responsive to pH changes compared with other receptor family members.11 Very recently, G2A was reported to be a receptor for 9-S-hydroxyeicosadienoic acid (9SHODE), a product of the 12/15-lipoxygenase family of enzymes.13 Although these data do not include direct binding, Obinata et al demonstrated that micromolar concentrations of 9SHODE induced G2A-dependent intracellular calcium mobilization, guanosine 5'-3-O-(thio)triphosphate binding, and pertussis toxin–sensitive inhibition of cAMP production.13 Thus, G2A is activated by multiple biological factors and may even serve different functions based on the specificity of these factors.
G2A is highly expressed on T cells, B cells, and macrophages, and lower expression levels have been found on endothelium.14 G2A has been localized to atherosclerotic plaques in the vessel wall of mice, suggesting a possible role for G2A in modulating atherosclerosis.15 In the current study, we examined the role of G2A in modulating endothelial activation and monocyte/endothelial interactions in vivo using G2A–/– mice. We report that G2A deficiency results in endothelial activation and increased monocyte adhesion to the vessel wall. Thus, we provide the first evidence of an antiinflammatory role of the G2A receptor in endothelium to protect against early events of atherogenesis.
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Materials and Methods |
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Detailed methods and reagents used can be found in the online data supplement, available at http://circres.ahajournals.org. C57BL/6 (B6) mice were purchased from The Jackson Laboratories. G2A–/– were obtained from Dr Owen Witte (University of California, Los Angeles). All animal studies were performed following approved guidelines of the University of Virginia Animal Care and Use Committee. B6 or G2A–/– mouse aortas were removed, opened longitudinally, pinned on agar, and used in an ex vivo monocyte adhesion assay as previously described.16 B6 and G2A–/– mice were injected with peritoneal macrophages from CX3CR1-GFP macrophages to measure trafficking to the aorta. Alternatively, aortic endothelial cells (ECs) were freshly harvested and cultured. Passage 2 ECs were used for conventional RT-PCR to measure G2A expression, and flow cytometry was performed to measure adhesion molecule expression. Monocyte adhesion to cultured ECs was performed using a flow chamber (Glycotech). Fluorescent microscopy for nuclear factor
B (NFB) was performed as described.17 Immunoblotting for G2A and signaling molecules was performed on protein extracts isolated from cultured ECs.16,17 |
Results |
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G2A–/– Aorta Binds More Monocytes Than Control Mouse Aorta
We directly tested whether monocyte adhesion to aortic endothelium was altered in G2A–/– mice versus control B6 mice. Aortas from G2A–/– and B6 mice were isolated and immediately used in an ex vivo monocyte adhesion assay. The WEHI 78/24 mouse monocyte cell line used in this assay has comparable G2A expression levels as primary B6 mouse monocytes (data not shown). Aortas from G2A–/– mice bound approximately 10-fold more monocytes than did B6 aortas (P<0.005) (Figure 1), suggesting that the aortic endothelium of the G2A–/– mice is highly activated. We hypothesized that monocyte/macrophage adhesion and accumulation in G2A–/– aortas would be similarly increased in vivo. To test this, we isolated peritoneal macrophages from heterozygous CX3CR1-GFP mice that express green fluorescent protein (GFP) in monocyte/macrophages.18 G2A expression in heterozygous CX3CR1-GFP macrophages was the same as in B6 macrophages (data not shown). The GFP-expressing macrophages were injected into G2A–/– and B6 recipient mice. After 96 hours, we isolated aortas and measured GFP expression using both immunohistochemistry and immunoblotting. Aortic root sections from B6 and G2A–/– mice were stained using an anti-GFP antibody and are shown in Figure 2A. GFP expression is significantly higher in the vessel wall of G2A–/– mice than B6 mice (Figure 2A). Moreover, homogenized aortas from G2A–/– mice exhibited greater expression of GFP (Figure 2B) than did B6 control mice by immunoblotting. These data indicate that G2A-deficient aortas accumulate more macrophages than B6 aortas. To determine whether there were differences in resident leukocyte populations in C57Bl/6J and G2A–/– aortas, we performed whole-aorta flow cytometry analysis, a technique developed by Galkina et al.19 There were significantly fewer T lymphocytes (CD3+) and significantly higher numbers of macrophages (CD11b+) in G2A–/– aortas compared with B6 aortas (Figure 2C). Thus, using 2 separate approaches, we have determined that there is greater macrophage accumulation in the aortic wall of G2A–/– mice compared with B6 control mice.
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We hypothesized that increased monocyte/endothelial interactions in G2A–/– aortas are caused by G2A deficiency in the endothelium rather than by G2A deficiency in resident leukocytes. To test this, we performed bone marrow transplant studies using bone marrow isolated from both C57Bl/6J mice and G2A–/– mice injected into 
-irradiated B6 and G2A–/– recipient mice. G2A–/– aortas bound significantly more monocytes in an ex vivo monocyte adhesion assay than B6 aortas (Figure 2D). This increase in monocyte adhesion to G2A–/– aortas appeared to be independent of whether the mice received bone marrow from C57Bl/6J or G2A–/– donors. These studies indicate that G2A deficiency in the endothelium is directly responsible for the increased adherence and accumulation of monocyte/macrophages to G2A–/– aortas in vivo.
Expression of Adhesion Molecules and Inflammatory Cytokines Is Upregulated in G2A–/– Endothelium
To understand the mechanisms contributing to enhanced monocyte adhesion to G2A–/– aortas, we isolated aortic endothelial cells from G2A–/– and control B6 mice. We have successfully isolated and characterized mouse aortic ECs (MAECs), as reported previously.20 Primary MAECs were used at passages 2 to 3, and all MAEC experiments were performed in DMEM containing 1% heat-inactivated FBS.
We observed a 3-fold increase in plasma levels of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 in G2A–/– mice (Figure 3A). Furthermore, IL-6 and MCP-1 mRNA, as well as VCAM-1 and ICAM-1 mRNA, levels were significantly induced in G2A–/– MAECs compared with B6 control (Figure 3B). IL-6 and MCP-1 are important in monocyte recruitment, and VCAM-1 and ICAM-1 play significant roles in monocyte adhesion to endothelium. Taken together, these data indicate that endothelial cells deficient in G2A display upregulation of adhesion molecules and induction of chemokine secretion, both of which will impact monocyte/endothelial adhesion. These data collectively suggest that the G2A receptor serves a protective, or antiinflammatory, role in aortic endothelium, and absence of G2A promotes endothelial activation.
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NFB Activation in G2A–/– Endothelium
VCAM-1, ICAM-1, IL-6, and MCP-1 have all been shown to be induced by nuclear factor B (NFB).21–24 NFB normally resides in the cytoplasm, where it is bound to inhibitory B (IB) and thereby inactive. For NFB activation to occur, the IB kinase (IKK) complex phosphorylates IB, causing rapid degradation of IB, thereby allowing NFB to translocate to the nucleus to initiate inflammatory gene transcription. Using total cell protein, we observed increases in mitogen-activated protein kinase kinase kinase (MEKK)-3, IKK, and IKKß (kinases responsible for the phosphorylation and subsequent degradation of IB) in G2A–/– ECs compared with B6 (Figure 4). We examined NFB activation in G2A–/– ECs through analysis of cytosolic and nuclear proteins isolated from G2A–/– and B6 ECs. Using immunoblotting for the p65 subunit of NFB and IB, we observed a significant 55% increase in NFB p65 expression in the nucleus and a corresponding 60% decrease in IB in the cytosol in G2A–/– ECs (Figure 4) compared with B6 ECs. These data implicate NFB as the mediator of the observed activation in G2A–/– ECs.
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"Add-Back" of G2A to G2A–/– Mouse Aortic Endothelium Reduces NFB Activation
To directly test the role of G2A in modulating endothelial activation and NFB, we performed experiments to restore, or "add-back," G2A to knockout cells. We transfected primary aortic ECs isolated from G2A–/– mice with a murine G2A expression plasmid to restore G2A expression in G2A–/– ECs. After transfection, we isolated total RNA from G2A–/– and B6 ECs and measured G2A mRNA expression. Transfection efficiency for MAECs was 60% to 65%, as determined by expression of a GFP reporter plasmid (Figure 5A). We wanted to restore G2A levels to those levels observed in B6 control mice rather than to achieve high levels of G2A expression in ECs. Transfection of ECs with 500 ng of G2A plasmid (G2ApEXV3) resulted in G2A mRNA levels (Figure 5B) and protein levels (Figure 5C) in ECs that approximated what was observed in control B6 mice. The control vector (+pEXV3) showed no change in G2A mRNA or protein expression (Figure 5).
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We directly tested the role of G2A in modulating NFB activation in aortic ECs. After transfection with murine G2A plasmid, ECs were stained for NFB p65 using an Alexa 488–conjugated anti-p65 antibody, and cells were visualized for nuclear p65 localization by fluorescent microscopy. The number of cells that were positive for nuclear p65 was counted. As shown in Figure 6, G2A–/– ECs had significantly more cells that were positive for nuclear NFB p65 (approximately 60% of cells) than did B6 ECs (approximately 15% of cells). Transfection of G2A–/– ECs with the pEXV3 control plasmid had no effect on NFB p65 localization. Addition of G2A to G2A–/– ECs reduced the number of cells positive for nuclear p65 to equal approximately 35% of cells (Figure 6). This is equivalent to a 55% reduction in the number of cells that are positive for nuclear p65, thereby indicating that NFB activation in aortic endothelium is regulated by endothelial G2A expression. Similar results were observed using an NFB-luciferase reporter assay (Figure 6).
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G2A Restoration Reduces Adhesion Molecule Expression on G2A–/– Endothelium
We next tested whether restoration of normal G2A expression in G2A–/– cells would modulate adhesion molecule expression. We transfected G2A–/– ECs with the murine G2A plasmid and analyzed adhesion molecule expression by flow cytometry. The surface expression of ICAM-1, VCAM-1, and E-selectin were all significantly higher on G2A–/– ECs than B6 ECs (Figure 7A). Addition of G2A back to G2A–/– ECs significantly reduced surface expression of these molecules (Figure 7A). There was no observed difference in P-selectin levels.
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G2A Add-Back Reduces Inflammatory Cytokine Expression in G2A–/– Endothelium
ELISAs were performed to measure the secretion of IL-6, keratinocyte-derived chemokine (KC), and MCP-1 from G2A–/– ECs, B6 ECs, and G2A–/– ECs in which G2A expression had been restored. Addition of G2A to G2A–/– ECs significantly reduced the production of these cytokines to control levels compared with G2A–/– ECs (Figure 7B). G2A–/– ECs were also transfected with a plasmid expressing a "nonphosphorylatable" form of IB, termed "super-repressor IB" (SR-IB). Expression of SR-IB sequesters NFB in the cytosol, thereby preventing NFB-dependent gene expression. Transfection of G2A–/– with the super-repressor IB construct reduced IL-6, KC, and MCP-1 to control levels, suggesting production of these chemokines in G2A–/– endothelium is NFB mediated. Transfection of B6 endothelial cells with the super-repressor IB plasmid had no significant effect on IL-6, KC, or MCP-1 secretion (data not shown).
RhoA Is Activated in G2A–/– Endothelium
RhoA has been shown to be an important mediator of NFB activation in endothelial cells.23 RhoA-dependent cytoskeletal rearrangement has been reported in mouse fibroblasts overexpressing G2A.25 In the current study, RhoA activation was measured using the G-LISA assay from Cytoskeleton. Interestingly, there was significantly higher RhoA activity (approximately 3-fold) in G2A–/– ECs compared with B6. Restoration of G2A expression in G2A–/– ECs resulted in significant reduction of RhoA activity (Figure 8A).
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Restoration of G2A Expression in ECs Reduces Monocyte Adhesion to Endothelium
Finally we tested whether G2A addition to G2A–/– ECs would modulate monocyte/endothelial interactions. B6, G2A–/–, and G2A–/– ECs transfected with control pEXV3 or G2ApEXV3 were used in a flow chamber system. WEHI 78/24 monocytes were flowed across the endothelium at a flow rate of 0.75 dyne/cm2. The numbers of firmly adherent WEHI monocytes to endothelium after 5 minutes were counted. As shown in Figure 8B, G2A–/– ECs display a 10-fold increase in the number of adherent monocytes compared with B6 control ECs. G2A add-back to G2A–/– ECs markedly reduces adhesion to endothelium by 80% (Figure 8B). Inhibition of NFB activation using the super-repressor IB plasmid reduced monocyte adhesion to G2A–/– endothelium by 90%, as did use of the pharmacological inhibitor of NFB, BAY 11-7085 (Figure 8B). Rho kinase inhibition using Y27632 significantly reduced monocyte adhesion to G2A–/– ECs. Neither Y27632 nor the super-repressor IB plasmid, nor the BAY compound, impacted monocyte adhesion to B6 control endothelium. These data indicate that G2A expression directly influences monocyte/endothelial interactions, most likely through reducing endothelial activation via suppression of NFB activation.
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Discussion |
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G2A is highly expressed on macrophages and lymphocytes and has been implicated in atherosclerosis.14,15 We identified the presence of G2A mRNA and protein in C57Bl/6J mouse aortic endothelial cells. The data from our current study indicate that endothelial expression of G2A serves a protective role in the aortic wall to prevent monocyte/endothelial interactions. These data provide the first evidence that G2A functions to regulate endothelial activation.
The notion that endothelial G2A expression reduces monocyte/endothelial interactions in the vessel wall in vivo is strengthened by the data shown in Figure 2. Figure 2 illustrates that macrophage accumulation in the aortic wall is significantly increased in G2A–/– recipient mice. In the first set of experiments, macrophages expressing GFP were isolated from heterozygous CX3CR1-GFP mice and reinjected into B6 control and G2A–/– recipients. The important concept in this experimental design is that the macrophages are from the same source; thus, the observed changes are not attributable to differences in macrophages. Immunostaining for GFP was performed in aortic sections. We observed more GFP in G2A–/– mice compared with B6 mice. Thus, G2A deficiency in the aortic wall contributes significantly to the enhanced macrophage accumulation in the vessel wall of G2A–/– mice. The second experiment shown in Figure 2 illustrates that the aortic wall of G2A–/– mice contains more macrophages than B6 control mouse aortas. Using a novel flow cytometry technique, we quantified the aortic wall leukocyte content in B6 versus G2A–/– mice. We found a significant increase in the number of macrophages present in the aortic wall of G2A–/– mice. We found no changes in B-cell content in the aortic wall, yet T-lymphocyte numbers were lower in G2A–/– aortas than in B6 controls. These data do not rule out potential differences in trafficking of the leukocytes to the aortic wall in the recipient animals, but trafficking of leukocytes to the aortic wall could be dramatically impacted by the endothelial activation state of the aortas. These data also do not rule out some contribution of leukocyte activation on the endothelial activation state in the G2A–/– mice. In an attempt to address such concerns, we performed a series of bone marrow transplant studies in which we examined monocyte adhesion to the aortic wall as a functional measure of endothelial activation. In this study, B6 or G2A–/– recipients were transplanted with either G2A–/– or B6 bone marrow. After 6 weeks of recovery on a rodent chow diet, aortas were harvested from the mice and used in an ex vivo assay to measure monocyte adhesion. If endothelial G2A expression were of primary importance in regulating monocyte/endothelial interactions, then we would have expected to find that G2A expression in myeloid cells would have no impact on monocyte adhesion to aorta. The results of the study showed exactly these results (Figure 2D), indicating that G2A expression in endothelium, not in myeloid cells, regulates monocyte/endothelial interactions in the aortic wall. The fact that we observed decreased T-lymphocyte accumulation in the wall suggests that lymphocyte activation of the endothelium may not be important in this particular case; however, it is possible that the lymphocytes that are present in the aortic wall contribute to endothelial activation. Both macrophages and lymphocytes in the aortic wall may indeed contribute to endothelial activation in the G2A–/– mice through cytokine secretion; however, our data suggest that the absence of G2A in endothelium is indeed a direct, significant contributor to endothelial activation.
With regard to signaling pathways in endothelium that may be regulated by G2A, the most striking evidence is the enhanced NFB activation observed in G2A–/– mice. We observed dramatic changes in nuclear translocation of NFB and in cytosolic IB degradation in G2A–/– endothelium (Figures 4 and 6
). Restoration of G2A expression strikingly reduced NFB activation, reduced endothelial expression of ICAM-1, IL-6, KC, and MCP-1, and dramatically reduced monocyte adhesion to endothelium. In contrast, Lin and Ye reported that overexpression of G2A in HeLa cells increased NFB-luciferase production.26 However, these studies were performed using overexpression of G2A in a HeLa cell line and transfection of RGS (regulator of G protein signaling) constructs to block NFB signaling. Although these overexpression studies suggest that NFB can be activated by G2A in a LPC-independent manner, they do not preclude that there is ligand-dependent NFB inhibition occurring in the cell in vivo. Perhaps control of NFB activation by G2A depends on ligand availability, or there could be cell-type specific. Our data indicate that NFB regulation by G2A is critical in endothelium. Recently, we observed upregulation of p38 mitogen-activated protein kinase and activator protein-1 in G2A–/– ECs using a set of gene arrays (data not shown). The p38 mitogen-activated protein kinase pathway is linked to NFB activation.27 Studies are currently ongoing in our laboratory to study these additional signaling pathways induced by G2A deficiency in aortic endothelium.
Despite conflicting evidence regarding the agonist/antagonist effects of LPC on G2A,7–10 LPC has remained a focal point in the studies of the role of G2A in atherosclerosis.14,15 Although direct binding of LPC to G2A is unlikely,8 there is evidence of indirect action of LPC on G2A cellular distribution.9 Recent studies in lipid metabolism28 suggest there is a link between LPC and arachidonic acid synthesis. The 12/15 lipoxygenase pathway converts arachidonic acid into 12-S-hydroxyeicosatetraenoic acid (12-S-HETE), 15-S-HETE, and lipoxin A4, all of which have been implicated in either a proinflammatory or antiinflammatory manner in atherosclerosis development.29–31 Obinata et al identifies 9-hydroxyoctadecadienoic acid (9SHODE), and other eicosanoids, as ligands for G2A,13 suggesting that G2A may be a multiligand receptor. Studies are currently ongoing in the laboratory to further identify eicosanoid ligands of G2A.
Despite evidence suggesting that G2A is activated by multiple lipids, there is also evidence to suggest that G2A is constitutively active in the absence of ligand. When overexpressed in fibroblasts, G2A causes constitutive RhoA activation through G13.25 Lin and Ye have reported that G2A stimulates the accumulation of both inositol phosphates and cAMP in the absence of ligand and that G2A differentially couples to Gs, Gq, or G13 depending on whether it is bound to ligand.26 Thus it is also possible that G2A is constitutively active in the cell, even in the absence of ligand.
In summary, we report that absence of G2A in aortic endothelium promotes endothelial activation and monocyte/endothelial interactions. Absence of G2A expression in endothelium promotes NFB activation and induces expression of proinflammatory endothelial chemokines and adhesion molecules. Thus, G2A expression in vascular endothelium may serve a protective role for prevention of early events of inflammation and atherosclerosis.
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Acknowledgments |
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We thank Dr Owen Witte and Dr Caius Radu (Howard Hughes Medical Institute, University of California, Los Angeles) for the gifts of the G2A–/– mice and murine G2A antibody. We thank Dr Steffen Jung (Weizmann Institute, Rehovot, Israel) and Dr Klaus Ley (University of Virginia) for the gift of the CX3CR1-GFP mice. We thank Dr Janusz Kabarowski (University of Alabama, Birmingham) for the gift of the control and murine G2A pEXV3 plasmids and Dr Dean Ballard (Vanderbilt University, Nashville, Tenn) for the super-repressor I
B plasmid. We thank Leah Cochran for assistance with mouse genotyping. We also thank Dr Klaus Ley and Dr Owen Witte for helpful discussions and critical review of the manuscript. Sources of Funding
This work was supported by NIH grant R01 HL071141 (to C.C.H.).
Disclosures
None.
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Footnotes |
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Original received July 29, 2006; revision received January 5, 2007; accepted January 12, 2007.
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References |
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