Interaction Between the Angiotensin-(1–7) Mas Receptor and the Dopamine D2 Receptor
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Abstract
Ang (angiotensin) 1–7 MasR (Mas receptor) and D2R (dopamine D2 receptor) stimulation is coupled to anti-inflammatory responses. In the present work, we investigated the hypothesis that the anti-inflammatory action mediated by both receptors results from MasR-D2R heteromerization. Human monocyte (THP-1) cells differentiated to macrophages and exposed to lipopolysaccharide were employed. Ang (1–7) and the D2R agonist SUM (sumanirole) induced a decrease in proinflammatory IL (interleukin) 6 release in human macrophages exposed to a proinflammatory stimulus. The Ang (1–7)–induced decrease in IL-6 was blocked by the D2R antagonist. Conversely, the SUM induced decrease in IL-6 was prevented by the MasR antagonist and when MasR expression was downregulated, suggesting MasR-D2R interaction. Co-immunoprecipitation assay in THP-1 cells and in human monocyte differentiated macrophages from peripheral blood mononuclear cells confirmed MasR-D2R interaction. To avoid the influence from other receptors, MasR-D2R interaction was characterized in transfected human embryonic kidney 293T cells. Fluorescence resonance energy transfer analysis showed that MasR and D2R formed a constitutive heteromer, which was not modified by their agonists. Ang (1–7) and dopamine stimulated ERK (extracellular signal-regulated kinase) 1/2 and Akt (protein kinase B) phosphorylation only in cells expressing MasR-D2R heteromers, but not in cells expressing each receptor alone. Ang (1–7)–stimulated ERK1/2 and Akt phosphorylation was prevented by D2R blockade while the effect of dopamine was prevented by MasR blockade, reinforcing the fact that MasR-D2R heteromers are involved in ERK1/2 and Akt activation induced by their agonists. Our findings provide new evidence regarding the mechanisms underlying the cross-talk between the Ang (1–7)/MasR axis and the dopaminergic system in response to a proinflammatory process.
Introduction
The renin-angiotensin system (RAS) and the dopaminergic system are both implicated in hypertension pathogenesis.1,2 The RAS is composed of 2 axes with opposing functions. The pressor axis, represented by AT1R (Ang [angiotensin] II type 1 receptor), mediates the vasoconstrictive, trophic, and proinflammatory effects of RAS.1 The depressor axis, on the other hand, exerts vasodilatory, anti-inflammatory, and antifibrotic effects through Ang (1–7) and its specific MasR (Mas receptor).1 MasR is constitutively coupled to multiple signaling pathways mediated by Gαs, Gαq, Gαi, and Gα12/13 proteins.1,3 Ang (1–7) through MasR stimulation elicits arachidonic acid release, nitric oxide generation and phospholipase A2, phosphatidylinositol 3-kinase/Akt (protein kinase B), MAPK (mitogen-activated protein kinase), and PKA (protein kinase A) activation.1,3
Dopamine receptors are classified into D1-like (D1 and D5) and D2-like (D2, D3, and D4) subtypes based on their structure and pharmacology.2 Besides its key role in cognition, mood, and motor movements, D2R (D2 receptor) stimulation is also involved in blood pressure regulation and in immune function.2 D2R stimulation leads to Gαi/o activation, resulting in adenylate cyclase inhibition, cAMP (cyclic adenosine monophosphate) reduction, and PKA inhibition.2
Inflammation is involved in hypertension pathogenesis.4 Cytokines, produced by activated immune cells acting as mediators of local inflammatory response, are implicated in the development and maintenance of hypertension.5 Both the RAS and the dopaminergic system influence the immune response.6,7 Evidence indicate a protective role both for Ang (1–7)/MasR and D2R agonists in regulating immune function through inhibition of proinflammatory cytokines secretion.2,8
A cross-talk between the RAS and the dopaminergic system has been shown, mainly in relation to renal and cardiovascular regulation.9 Both systems also interact in regulating the immune response.10 Dopamine modulates inflammation in substantia nigra and striatum via Ang II/AT1R downregulation.10 Despite the fact that renal physiological evidence suggests the existence of an interaction between Ang (1–7) and dopamine,11 consequences of this interaction on the immune response are still unknown. This interaction may take place at the receptor level.
GPCRs (G-protein coupled receptors) form homo- or hetero-oligomers showing distinct properties relative to the monomeric entities.12 GPCRs heteromerization modulates receptor biosynthesis, trafficking, ligand affinity, intracellular signaling, and their ultimate biological response.12 Both MasR and D2R form heteromers with other GPCRs.13–16 MasR-AT1R heteromers alter AT1R intracellular trafficking and decrease Ang II-induced production of inositol phosphate and intracellular Ca2+ mobilization in transfected cells.13 While MasR heteromerization with the bradykinin B2 receptor results in an increased ligand affinity and a delayed MasR sequestration from the plasma membrane,14 D2R heteromerization with AT1R is associated to attenuated Ca2+ and mitogenic response to Ang II.15 The existence of D2R-AT1R heteromers in rat striatum explains the ability of candesartan, a selective AT1R antagonist, to block the reduction in cAMP, MAPK activation, and β-arrestin recruitment induced by a D2R agonist.15 Taking into account that MasR and D2R are GPCRs, we hypothesize that the anti-inflammatory action of Ang (1–7) and dopamine results from MasR-D2R heteromerization. Our aim was to investigate MasR-D2R heteromerization and its involvement in the immune response elicited by their agonists.
Materials and Methods
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Cell Culture
Human monocytic leukemia (THP-1) cell line obtained from the American Type Culture Collection (United States) was grown in Roswell Park Memorial Institute 1640 culture medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mmol/L L-glutamine, at 37 °C in 5% CO2 in a humidified incubator. THP-1 cells were differentiated to macrophages in the presence of phorbol-12-myristate-13-acetate, acquiring a phenotype comparable to those of human peripheral blood mononuclear cell (PBMC)-derived macrophages.17 Human embryonic kidney (HEK) 293T cells obtained from American Type Culture Collection were grown in DMEM high-glucose supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2 in a humidified incubator.
PBMC Isolation, Monocyte Enrichment, and Differentiation to Macrophages
Monocyte differentiated macrophages from PBMC were obtained as previously described.18
Ligand Concentrations
Ang-(1-7) or dopamine (1 μmol/L) were employed in experiments performed with HEK293T cells and 100 nmol/L Ang (1–7) or SUM (sumanirole) (D2R agonist) were used in those performed with THP-1 cells. Those concentrations have been previously employed by us and other researchers14,19 and when immunomodulatory actions of SUM16 and Ang (1–7)20 were evaluated. More information is available in the Data Supplement.21–24
IL 6 Determination
IL (interleukin) 6 levels were measured in LPS (lipopolysaccharide)-stimulated THP-1 cells differentiated to macrophages by ELISA.
FRET by Acceptor Photobleaching
MasR-D2R heteromerization was investigated by fluorescence resonance energy transfer (FRET) after acceptor photobleaching as previously described.14
ERK and Akt Phosphorylation Assay
It was evaluated by Western blot as previously described.14
Co-Immunoprecipitation
MasR and D2R interaction was evaluated by co-immunoprecipitation as previously described.14
Detailed methods are available in the Data Supplement.
Statistical Analysis
Results are presented as mean±SEM. One-way ANOVA followed by Bonferroni post-test was used to analyze data with unequal variance between each group. A probability level of 0.05 was considered significant.
Results
Anti-Inflammatory Effects Elicited by Ang (1–7) and the D2R Agonist Are Mediated by MasR-D2R Interaction
First, we investigated whether MasR and D2R were expressed in human THP-1 macrophages. Figure S3 in the Data Supplement shows that both receptors were expressed in human macrophages. HEK293T cells transfected with MasR and D2R cDNA were used as positive controls.
The anti-inflammatory effect elicited by Ang (1–7) and the D2R specific agonist SUM was evaluated in LPS-stimulated THP-1 macrophages, a sensitive in vitro system previously employed to analyze immunomodulatory activity of different compounds.17 As it is shown in Figure 1, Ang (1–7) induced a decrease in IL-6 levels in LPS-stimulated THP-1 cells, which was partially blocked by the MasR antagonist A-779, demonstrating that the anti-inflammatory effect elicited by Ang (1–7) is mediated by MasR activation. To further confirm this result, the effect of Ang (1–7) on IL-6 secretion was evaluated in cells previously transfected with a specific siRNA targeted to MasR. Figure S4 shows that MasR expression was downregulated when a siRNA against MasR was employed. Under these conditions, Ang (1–7)–induced decrease in IL-6 levels was partially prevented (Figure 1). The diminished IL-6 release caused by Ang (1–7) was not modified in THP-1 cells transfected with a nontargeting siRNA (Figure S5). In addition, the reduction in IL-6 exerted by Ang (1–7) was also partially blocked by the specific D2R antagonist L-741,626 (Figure 1). Altogether these results demonstrate that the Ang (1–7)–induced decrease in IL-6 involves MasR and D2R, suggesting MasR-D2R interaction in the anti-inflammatory action of Ang (1–7).
Figure 1. The decrease in IL (interleukin)-6 induced by Ang (angiotensin) 1–7 was prevented when D2R (dopamine D2 receptor) was blocked. Human monocytic cells (THP-1) differentiated to macrophages were incubated for 24 h in the absence (basal) or presence of LPS (lipopolysaccharide), LPS+Ang (1–7), LPS+Ang (1–7)+MasR (Mas receptor) antagonist D-Ala7-Ang (1–7; A-779) or LPS+Ang (1–7)+D2R antagonist L-741,626. THP-1 macrophages previously transfected with siRNA targeted to MasR were incubated with Ang (1–7) under a proinflammatory stimulus with LPS. IL-6 levels were measured as described in Methods. No changes in IL-6 levels were seen after transfection with a nontargeting control siRNA in comparison to Ang (1–7) treatment (Figure S3). Values are expressed as the mean±SEM from 5 independent experiments. ANOVA with Bonferroni post hoc test was used for multiple comparisons. &P<0.0001 vs basal; $P<0.01 vs basal; #P<0.01 vs Ang (1–7); and *P<0.05 vs Ang (1–7).
The specific D2R agonist SUM induced a decrease in IL-6 secretion in THP-1 macrophages exposed to LPS and this effect was partially blocked by the D2R antagonist, evidencing that the anti-inflammatory effect elicited by SUM is coupled to D2R activation (Figure 2). The reduction in IL-6 exerted by SUM was partially blocked by the MasR antagonist, suggesting MasR-D2R interaction (Figure 2). To further confirm that MasR-D2R interaction is involved in the anti-inflammatory action of SUM, the effect of the D2R agonist was assayed in cells with MasR expression downregulated. As it is shown in Figure 2, when MasR was decreased by a siRNA targeted to MasR, the SUM-mediated decrease in IL-6 release was partially prevented, and it was not modified in cells transfected with a nontargeting siRNA (Figure S6), demonstrating that the anti-inflammatory effect of SUM is mediated by MasR-D2R interaction.
Figure 2. The decrease in IL (interleukin)-6 induced by SUM (sumanirole) was prevented when MasR (Mas receptor) was blocked or downregulated. Human monocytic cells (THP-1) differentiated to macrophages were incubated for 24 h in the absence (basal) or presence of LPS (lipopolysaccharide), LPS+SUM, LPS+SUM+D2R (dopamine D2 receptor) antagonist L-741,626 or LPS+SUM+MasR antagonist D-Ala7-Ang (angiotensin) 1–7; A-779). THP-1 macrophages previously transfected with siRNA targeted to MasR were incubated with SUM under a proinflammatory stimulus with LPS. IL-6 levels were measured as described in Methods. No changes in IL-6 levels were seen after transfection with a nontargeting control siRNA in comparison to SUM treatment (Figure S4). Values are expressed as the mean±SEM from 5 independent experiments. ANOVA with Bonferroni post hoc test was used for multiple comparisons. &P<0.0001 vs basal; $P<0.01 vs basal; #P<0.001 vs SUM; and *P<0.01 vs SUM.
Altogether, these results suggest MasR-D2R heteromerization in human macrophages.
D2R Interacts With MasR in THP-1 Macrophages
To further confirm the existence of an interaction between MasR and D2R in THP-1 macrophages, MasR-D2R co-immunoprecipitation was performed. Upon immunoprecipitation of MasR from THP-1 macrophages lysate co-precipitation of D2R was observed, confirming the existence of MasR-D2R interaction in human macrophages (Figure 3A).
Figure 3. MasR (Mas receptor) and D2R (dopamine D2 receptor) interact in THP-1 and in human monocyte-derived macrophages. Immunoprecipitation of lysates from human THP-1 macrophages (n=3; A) and human monocyte-derived macrophages from PBMC (n=2; B). Immunoprecipitates (IP) were analyzed by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. Representative blots with similar qualitative results are presented. MW indicates molecular weight; and NS, nonspecific, which refers to the use of control rabbit IgG instead of the primary antibody in the co-immunoprecipitation.
D2R Interacts With MasR Under Physiological Conditions
To establish that our results are not specific to THP-1 cells, we performed MasR-D2R co-immunoprecipitation in human monocyte differentiated macrophages from PBMC. Upon immunoprecipitation of D2R co-precipitation of MasR was observed (Figure 3B). Conversely, upon immunoprecipitation of MasR co-precipitation of D2R was observed (Figure 3B).
MasR-D2R Heteromerization in HEK293T Cells
To avoid the influence of other receptors present in THP-1 cells, MasR-D2R interaction and its functional consequences were investigated in transfected HEK293T cells. Interaction between MasR and D2R was evaluated by FRET measured by acceptor photobleaching (Figure 4A). Two fusion proteins were expressed: MasR-YFP (MasR fused to the yellow fluorescent protein) through its C-terminus and D2R-mCherry (D2R fused to mCherry fluorescent protein) through its C-terminus. A representative FRET experiment is shown in Figure 4B and 4C. Before photobleaching, YFP and mCherry fluorescence was observed at the cell membrane and cytoplasm of HEK293T cells transfected with MasR-YFP plus D2R-mCherry. After acceptor photobleaching, YFP fluorescence intensity increased if interaction between receptors occurs, resulting in positive FRET, thus of MasR-D2R heteromerization. To quantify FRET, the apparent FRET efficiency was calculated, which in cases of tight interactions is proportional to the fraction of the donor forming a complex with the acceptor. A mean apparent FRET efficiency of 0.22±0.01 was measured in cells transiently co-expressing MasR-YFP and D2R-mCherry with similar donor (YFP) to acceptor (mCherry) ratio (Figure 4D). These results demonstrate that both MasR and D2R constitutively interact in HEK293T cells by a heteromer formation.
Figure 4. MasR (Mas receptor) and D2R (dopamine [DA] D2 receptor) form a constitutive heteromer in HEK293T cells.A, Schematic representation of fluorescence resonance energy transfer (FRET). MasR fused to EYFP (enhanced yellow fluorescence protein) (FRET donor) and D2R fused to mCherry (FRET acceptor) are co-expressed in the same cell. Upon excitation of the donor molecule not only the donor emits the energy (light gray arrow), but also part of the energy is transferred to the acceptor which also emits (dark gray arrows; top). Photodestruction of the acceptor molecule abolishes energy transfer, evidenced by an increase in the donor emission (light gray arrows; bottom). B, Representative images of prebleaching and postbleaching of cells expressing MasR-YFP (yellow fluorescent protein; FRET donor) and D2R-mCherry (FRET acceptor). Scale bar: 2.5 μm. C, Representative plot of fluorescence intensity of YFP and mCherry along a horizontal line drawn across the cytoplasm of cells expressing MasR-YFP+D2R-mCherry prebleaching and postbleaching. Quantification of fluorescence intensity was performed using Image J analyzer software (RSB); au indicates arbitrary units. D, Apparent FRET efficiency measured in cells transfected with the empty vector (mocked) or the vectors containing cDNA coding for MasR-YFP+D2R-mCherry incubated in the absence (basal) or presence of 1 μmol/L Ang (angiotensin)-(1–7) or dopamine for 15 min at 37 °C. Values are expressed as the mean±SEM from 4 independent experiments. ANOVA with Bonferroni post hoc test was used for multiple comparisons. Twenty-five cells were analyzed per experiment. *P<0.001 vs mocked. E, Immunoprecipitation of lysates from MasR+D2R transfected HEK293T cells was performed using (1) anti-D2R or (2) anti-MasR antibodies. Immunoprecipitates (IP) were analyzed by SDS-PAGE and immunoblotted (IB) using the indicated antibodies. Representative blots of 2 different experiments with similar qualitative results are presented. MW indicates molecular weight; and NS, nonspecific, which refers to omission of the primary antibody in the co-immunoprecipitation assay.
Agonists influence on MasR-D2R heteromerization was studied in HEK293T cells transiently expressing MasR-YFP plus D2R-mCherry and incubated with 1 μmol/L Ang (1–7) or dopamine for 15 minutes at 37 °C. As shown in Figure 4D, FRET was not modified when the cells were stimulated with either of the agonists, indicating that MasR-D2R heteromer formation was not influenced by Ang (1–7) or dopamine. Thus, MasR-D2R heteromerization is constitutive and not altered by their agonists.
To further confirm MasR-D2R heteromerization, co-immunoprecipitation of both receptors was performed in HEK293T cells co-transfected with the cDNA coding for MasR and D2R. Upon immunoprecipitation of D2R, co-precipitation of MasR was observed (Figure 4E). The presence of 2 bands may be due to post translational modifications such as glycosylation, as it has been previously suggested for this receptor.25 Conversely, upon immunoprecipitation of MasR co-precipitation of D2R was observed (Figure 4E). The presence of higher molecular weight bands for D2R may be due to the existence of different forms of glycosylated receptor as suggested previously.26 To further confirm antibody specificity, immunoprecipitation of MasR in HEK293T cells expressing only D2R was performed. Under this condition, no band was detected (Figure S7).
Altogether, these results support the existence of constitutive MasR-D2R heteromerization.
MasR-D2R Heteromerization and Downstream Signaling
Heteromerization among GPCRs has been demonstrated to alter receptor signaling.12 MasR-D2R heteromerization on ERK (extracellular signal-regulated kinase) 1/2 MAPK or Akt activation was investigated. Ang (1–7) or dopamine had no effect on ERK1/2 phosphorylation in cells transiently expressing MasR or D2R alone, respectively (Figure 5A). Cells transiently co-expressing both MasR and D2R did not show changes in ERK1/2 phosphorylation under basal conditions compared with cells expressing only one of the receptors (Figure 5B), evidencing that basal ERK1/2 activation is not modified by MasR-D2R heteromerization. Conversely, both Ang (1–7) and dopamine promoted ERK1/2 activation in cells co-expressing both MasR and D2R (Figure 5C). The effect of Ang (1–7) was prevented by D2R blockade, while the effect of dopamine was prevented by MasR blockade (Figure 5C), reinforcing the concept of MasR-D2R interaction.
Figure 5. MasR (Mas receptor)-D2R (dopamine [DA] D2 receptor) heteromerization is involved in Ang (angiotensin) 1–7 or DA-mediated ERK (extracellular signal-regulated kinase) 1/2 activation. ERK1/2 phosphorylation in (A) HEK293T cells expressing MasR or D2R incubated in the absence (basal, B) or presence of Ang (1–7; Ang) or DA, respectively, in (B) HEK293T cells expressing MasR, D2R, or MasR+D2R under basal conditions or in (C) HEK293T cells expressing MasR+D2R incubated in the absence (basal) or presence of Ang (1–7; Ang) or Ang (1–7)+D2R antagonist L-741,626 or DA or DA+MasR antagonist A-779 for 15 min. Quantification of Western blots was performed using Image J analyzer software (RSB). Representative Western blots are shown above each graph. Results are expressed as fold change of the response detected in HEK293T cells expressing MasR in basal conditions. Each bar represents the mean±SEM of four independent preparations. ANOVA with Bonferroni post hoc test was used for multiple comparisons. *P<0.01 vs basal; &P<0.001 vs basal; #P<0.01 vs Ang (1–7); and $P<0.001 vs DA. p-ERK1/2 indicates phosphorylated ERK1/2
Regarding Akt activation, Ang (1–7) or dopamine had no effect on Akt phosphorylation in cells transiently expressing MasR or D2R alone, respectively (Figure 6A). Cells transiently co-expressing both MasR and D2R did not showed changes in Akt phosphorylation under basal conditions compared with cells expressing only one of the receptors (Figure 6B). In contrast, both Ang (1–7) and dopamine promoted Akt phosphorylation in cells co-expressing both MasR and D2R (Figure 6C). The effect of Ang (1–7) was prevented by D2R blockade while the effect of dopamine was prevented by MasR blockade (Figure 6C), supporting MasR-D2R heteromerization.
Figure 6. MasR (Mas receptor)-D2R (dopamine [DA] D2 receptor) heteromerization is involved in Ang (angiotensin) 1–7 or DA-mediated Akt (protein kinase B) activation. Akt phosphorylation in (A) HEK293T cells expressing MasR or D2R incubated in the absence (basal, B) or presence of Ang (1–7; Ang) or DA, respectively, in (B) HEK293T cells expressing MasR, D2R, or MasR+D2R under basal conditions or in (C) HEK293T cells expressing MasR+D2R incubated in the absence (basal) or presence of Ang (1–7; Ang) or Ang (1–7)+D2R antagonist L-741,626 or DA or DA+MasR antagonist A-779 for 15 min. Quantification of Western blots was performed using Image J analyzer software (RSB). Results are expressed as fold change of the response detected in HEK293T cells expressing MasR in basal conditions. Each bar represents the mean±SEM of 4 independent preparations. ANOVA with Bonferroni post hoc test was used for multiple comparisons. *P<0.01 vs basal; #P<0.05 vs Ang (1–7); and &P<0.01 vs DA. p-Akt indicates phosphorylated Akt.
Altogether, these results support the existence of MasR-D2R heteromers. MasR-D2R heteromerization is necessary for Ang (1–7) and dopamine to induce ERK1/2 and Akt activation.
Discussion
The present study showed for the first time the existence of MasR-D2R heteromerization which is constitutive and not influenced by their agonists. MasR-D2R heteromerization is involved in the anti-inflammatory action mediated by MasR or D2R stimulation in human macrophages. The results of co-immunoprecipitation experiments in human monocyte differentiated macrophages from PBMC provide direct evidence that these receptors also interact under physiological conditions (present results).
Our present study showed that Ang (1–7) induced a decrease in IL-6 in human macrophages exposed to an inflammatory stimulus. Accordingly, Ang (1–7), through MasR stimulation, reduced IL-6 expression in LPS-stimulated macrophages isolated from mice8 and AVE0991, an Ang (1–7) mimetic, inhibited proinflammatory cytokines secretion induced by LPS in THP-1 macrophages, effects partially reversed by a MasR antagonist.27 In fact, MasR deletion alters macrophage function and phenotype, aggravating the clinical course of disease in mouse models of autoimmune encephalomyelitis and atherosclerosis.28 Altogether these reports demonstrate a protective role for MasR in inflammation.
D2R stimulation is coupled to anti-inflammatory responses.29,30 D2R activation prevents the increased TNF-α (tumor necrosis factor α) induced by LPS in peritoneal macrophages harvested from Swiss mice29 and in bone-marrow macrophages from mice.30 Furthermore, the specific D2R agonist SUM induces a reduction in IL-6 release in endothelial cells subjected to a proinflammatory stimulus.16 Interestingly, the decrease in IL-6 induced by SUM in human macrophages was reduced when MasR was blocked or downregulated (present results) indicating that the D2R-mediated anti-inflammatory effect is mediated at least in part by MasR-D2R interaction. Conversely, the decrease in IL-6 secretion coupled to MasR stimulation was prevented when D2R was blocked reinforcing MasR-D2R interaction.
Evidence regarding a cross-talk between the dopaminergic system and the Ang (1–7)/MasR axis is scarce. Results from our laboratory showed that Ang (1–7)–induced inhibition of Na+, K+-ATPase activity is due to Ang (1–7) stimulation of renal extraneuronal uptake of dopamine,11 suggesting an interaction between both systems. Natriuretic Ang (1–7) effects were abolished by both MasR and D1-like receptor blockade.31 However, the effect of a selective D1-like receptor agonist was insensitive to A-779.31 These data suggest that the Ang (1–7)–mediated Na+ excretion, but not that from D1-like receptor stimulation, is mediated by MasR-D1-like receptor interaction. Because the authors did not investigate the putative heteromerization between MasR and D1R, we could not disregard that Ang (1–7) natriuretic response depends on MasR-D1R heteromerization. Heteromers may exhibit unidirectional cross-antagonism.
Our work showed that MasR or D2R antagonists failed to completely abolish the IL-6 response to Ang (1–7). In agreement, the decrease in proinflammatory cytokine secretion exerted by the Ang (1–7) mimetic AVE0991 in LPS-stimulated THP-1 macrophages has also been shown to be partially prevented by MasR blockade.27 A possible explanation for partial blockade of Ang (1–7) responses may be due to the involvement of other receptors in such action. We could not disregard that MrgDR, the receptor that mediates alamandine actions,32 may also be involved in the anti-inflammatory action of Ang (1–7) because it has been shown that this peptide recognizes not only MasR but also MrgDR.33 Unpublished results from our lab showed that Ang (1–7) binding to human THP-1 macrophages was displaced by D-Pro7-Ang (1–7), which is a MasR and MrgDR antagonist.33,34 Up to date, there is not a specific MrgDR antagonist. Consequently, the results obtained do not allow us to confirm or rule out the participation of MrgDR in the Ang (1–7) anti-inflammatory actions in THP-1 macrophages. Some Ang (1–7) responses are mediated partly through AT1R or AT2R,35 however, we disregard AT1R or AT2R involvement in the anti-inflammatory action of Ang (1–7), since specific Ang (1–7) binding to THP-1 cells was not displaced by increasing concentrations of losartan (AT1R antagonist), PD 123319 (AT2R antagonist) or compound 21 (AT2R agonist; unpublished results), excluding Ang (1–7) binding to AT1 or AT2 receptors. Another possibility is that MasR heteromerization with other receptors may also be involved in the anti-inflammatory action of Ang (1–7). Receptors are in constant dynamism, taking different conformations, interacting with other receptors or other proteins.12 Although other dopamine receptor subtypes may also be involved, we disregard D3R and D5R participation because both receptors promote inflammation.36 In contrast, D1R and D2R are coupled to anti-inflammatory mechanisms. Thus, D1R may also form an heteromer with MasR and in this way contribute to the anti-inflammatory action of Ang (1–7). Further experiments have to be done to test this hypothesis.
Heteromerization may result in changes in receptor signaling. AT1R-D2R heteromerization has been shown not to be influenced by their agonists, instead differences in signaling at different doses of the agonist were observed.15 In accordance, our study showed that concentration as high as 1 µmol/L did not modify MasR-D2R heteromerization, but induced changes in signaling. MasR-D2R heteromerization was involved in ERK1/2 and Akt activation induced by Ang (1–7) and dopamine (present results). Accordingly, dopamine elicited an increase in ERK1/2 phosphorylation only in HEK293 cells expressing the heteromer between D2R and the serotonin 5-HT1A receptor but not in cells expressing each one of the receptors separately.37 Thus, it seems that ERK1/2 activation mediated by D2R stimulation needs the interaction with other receptors. Ang (1–7) effects on ERK1/2 activation are variable and depend on the cell type involved.38–42 Ang (1–7) promoted ERK1/2 activation in MasR transfected HEK293T cells and in neurons from normotensive rats.38,39 In addition, Ang (1–7) through MasR up-regulates neuronal norepinephrine transporter via Akt and ERK1/2-dependent pathways in hypothalamic neuronal cultures from spontaneously hypertensive rats.40 In contrast, Ang (1–7) attenuated the Ang II-stimulated phosphorylation of ERK1/2 in proximal tubular cells41 and TGF-β (transforming growth factor-beta)-dependent stimulation of ERK1/2 pathway in proximal epithelial cells.42 All these findings are consistent with the notion that ERK1/2 (and the MAPK pathway) transduces a variety of extracellular signals, resulting in different biological responses.
Perspectives
In conclusion, we have demonstrated that MasR and D2R interact and form constitutive heteromers which are not altered by their agonists. MasR-D2R heteromerization is involved in the activation of ERK1/2 and Akt induced by Ang (1–7) and dopamine. Moreover, MasR-D2R interaction is implicated in the anti-inflammatory properties exerted by their agonists. MasR-D2R heteromerization and their functional consequences open our understanding of the mechanisms underlying the cross-talk between the Ang (1–7)/MasR axis of the RAS and the dopaminergic system, which have not yet been elucidated until today. The present work provides new evidence on the mechanism by which Ang (1–7) and dopamine exert an anti-inflammatory action in response to a proinflammatory process. Since inflammation is involved in hypertension pathogenesis, compounds targeting MasR-D2R heteromer could represent a promising pharmacological tool in the development of new drug candidates for cardiovascular diseases treatment.
Nonstandard Abbreviation and Acronyms
| Akt | protein kinase B |
| Ang II | angiotensin II |
| AT1R | Ang II type 1 receptor |
| cAMP | cyclic adenosine monophosphate |
| D2R | D2 receptor |
| D2R-mCherry | D2R fused to mCherry fluorescent protein |
| ERK | extracellular signal-regulated kinase |
| FRET | fluorescence resonance energy transfer |
| GPCR | G-protein coupled receptor |
| HEK | human embryonic kidney |
| IL-6 | interleukin 6 |
| LPS | lipopolysaccharide |
| MAPK | mitogen-activated protein kinase |
| MasR | receptor Mas |
| MasR-YFP | MasR fused to the yellow fluorescent protein |
| PBMC | peripheral blood mononuclear cell |
| PKA | protein kinase A |
| RAS | renin-angiotensin system |
| SUM | sumanirole |
| TGF-β | transforming growth factor-beta |
| TNF-α | tumor necrosis factor α |
Acknowledgments
We thank Josefina Brunori for her assistance in the preparation of the final artwork and figure formatting.
Sources of Funding
This work was supported by grants from Universidad de Buenos Aires [20020160100134BA] and Agencia Nacional de Promoción Científica y Tecnológica [PICT 2016-2978, PICT-2019-2384].
Disclosures None.
Footnotes
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Novelty and Significance
What Is New?
Both MasR (Mas receptor)
and D2R (dopamine D2 receptor)
exert anti-inflammatory responses. We provide evidence for the first time that MasR forms heteromeric complexes with D2R. MasR-D2R heteromerization is involved in the anti-inflammatory effect induced by MasR and D2R stimulation in human macrophages.
What Is Relevant?
MasR-D2R heteromerization is implicated in the anti-inflammatory properties exerted by their agonists in human macrophages.
Summary
MasR forms heteromeric complexes with D2R on the cell surface. MasR-D2R heteromerization is constitutive and not altered by their agonists. MasR-D2R heteromers are involved in ERK (extracellular signal-regulated kinase) 1/2 and Akt (protein kinase B) activation induced by Ang (angiotensin)-1–7 and dopamine. The interaction between MasR-D2R also mediates the anti-inflammatory effects exerted by their agonists in human macrophages. These findings provide new evidence regarding the immunomodulatory effects of Ang (1–7) and dopamine in a proinflammatory process which has been linked to the pathogenesis of hypertension. Compounds targeting MasR-D2R heteromer could represent a promising pharmacological tool in the development of new drug candidates for cardiovascular diseases treatment.
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