ORIGINAL RESEARCH published: 27 May 2021 doi: 10.3389/fendo.2021.635923 Crosstalk Between LXR and Caveolin-1 Signaling Supports Cholesterol Efflux and Anti-Inflammatory Pathways in Macrophages Cristina M. Ramı́rez 1*, Marta Torrecilla-Parra 1, Virginia Pardo-Marqués 1, Edited by: Mario Fernández de-Frutos 1, Ana Pérez-Garcı́a 1, Carlos Tabraue 2,3, Ines Pineda-Torra, Juan Vladimir de la Rosa 2, Patricia Martı́n-Rodriguez 2, Mercedes Dı́az-Sarmiento 2, University College London, Uxue Nuñez 2, Marta C. Orizaola 4, Paqui G. Través 4, Marta Camps 5, Lisardo Boscá 2,4,6 United Kingdom and Antonio Castrillo 2,4* Reviewed by: 1 Instituto Madrileño de Estudios Avanzados (IMDEA) Research Institute of Food and Health Sciences, Madrid, Spain, Peter Tontonoz, 2 Unidad de Biomedicina (Unidad Asociada al CSIC), Instituto Universitario de Investigaciones Biomédicas y Sanitarias UCLA Health System, United States (IUIBS) de la Universidad de Las Palmas de Gran Canaria, Las Palmas, Spain, 3 Departamento de Morfologı´a, Universidad Edgar Zenteno, de Las Palmas de Gran Canaria, Las Palmas, Spain, 4 Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo National Autonomous Superior de Investigaciones Cientı´ficas CSIC-Universidad Autónoma de Madrid, Madrid, Spain, 5 Institut de Biomedicina University of Mexico, Mexico (IBUB), Universitat de Barcelona, Barcelona, Spain, 6 Centro de Investigación en Red sobre Enfermedades Cardiovasculares *Correspondence: (CIBERCV), Madrid, Spain Antonio Castrillo acastrillo@iib.uam.es Cristina M. Ramı´rez Macrophages are immune cells that play crucial roles in host defense against pathogens cristina.ramirez@imdea.org by triggering their exceptional phagocytic and inflammatory functions. Macrophages that Specialty section: reside in healthy tissues also accomplish important tasks to preserve organ homeostasis, This article was submitted to including lipid uptake/efflux or apoptotic-cell clearance. Both homeostatic and Molecular and Structural inflammatory functions of macrophages require the precise stability of lipid-rich Endocrinology, a section of the journal microdomains located at the cell membrane for the initiation of downstream signaling Frontiers in Endocrinology cascades. Caveolin-1 (Cav-1) is the main protein responsible for the biogenesis of Received: 30 November 2020 caveolae and plays an important role in vascular inflammation and atherosclerosis. The Accepted: 24 February 2021 Published: 27 May 2021 Liver X receptors (LXRs) are key transcription factors for cholesterol efflux and Citation: inflammatory gene responses in macrophages. Although the role of Cav-1 in cellular Ramı´rez CM, Torrecilla-Parra M, cholesterol homeostasis and vascular inflammation has been reported, the connection Pardo-Marqués V, de-Frutos MF, between LXR transcriptional activity and Cav-1 expression and function in macrophages Pérez-Garcı´a A, Tabraue C, de la Rosa JV, Martı´n-Rodriguez P, has not been investigated. Here, using gain and loss of function approaches, we Dı´az-Sarmiento M, Nuñez U, demonstrate that LXR-dependent transcriptional pathways modulate Cav-1 expression Orizaola MC, Través PG, Camps M, Boscá L and Castrillo A (2021) and compartmentation within the membrane during macrophage activation. As a result, Crosstalk Between LXR and Cav-1 participates in LXR-dependent cholesterol efflux and the control of inflammatory Caveolin-1 Signaling Supports responses. Together, our data show modulation of the LXR-Cav-1 axis could be exploited Cholesterol Efflux and Anti-Inflammatory Pathways to control exacerbated inflammation and cholesterol overload in the macrophage during in Macrophages. the pathogenesis of lipid and immune disorders, such as atherosclerosis. Front. Endocrinol. 12:635923. doi: 10.3389/fendo.2021.635923 Keywords: gene expression, caveolin-1, cholesterol efflux, inflammation, LXR, macrophage Frontiers in Endocrinology | www.frontiersin.org 1 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages INTRODUCTION functional in different immune cells, including macrophages (20–26). Nevertheless, although several studies in the last two Liver X Receptors (LXRa and LXRb) are transcription factors that decades reported different roles for Cav-1 in the context of belong to the nuclear receptor superfamily. These are endogenously macrophage biology, the role of Cav-1 in macrophages is not activated by oxidized forms of cholesterol (oxysterols), and function completely understood. In these cells, Cav-1 appears to as intracellular sensors of cholesterol levels (1). The accumulation of participate in apoptosis, lipid and cholesterol metabolism, as cholesterol in macrophages, derived from the uptake of lipoproteins well as an anti-inflammatory mediator (27–29). Importantly, or cellular debris, leads to LXR activation and triggers the induction of Cav-1 expression has been shown to increase in response to LPS a transcriptional program to promote cholesterol utilization. One of but the overall participation of this protein in immune processes these pathways promotes the export of cholesterol and phospholipids is not entirely clear (30, 31). Since dysregulation of lipid and outside the cell, through the transcriptional induction of members of immune homeostasis in macrophages are contributing factors the ATP binding cassette family such as ABCA1 and ABCG1 (2, 3). for the development of several chronic diseases, our study aims Indeed, the capacity of LXR synthetic ligands to inhibit the to explore the possible participation of Cav-1 in the metabolic development of atherosclerosis in mice results, in part, by and inflammatory effects of LXR in macrophages. promoting the exit of excess cholesterol from lipid loaded macrophages or foam cells, known as cholesterol efflux (4). In addition to their important role in cholesterol metabolism, LXRs MATERIAL AND METHODS are also involved in inflammation and in the regulation of immune responses. Activation of LXR with synthetic ligands has been shown Animal Procedures to repress the expression of inflammatory genes in macrophages such Cav-1 deficient mice (Cav-1−/−, strain Cav-1tm1Mls/J, genetic as inducible nitric oxide synthase (iNOS), COX-2 or pro- background 129/Sv, C57BL/6J, and SJL) and their corresponding inflammatory cytokines including interleukin-6 (IL-6) and controls Cav‐1+/+ were obtained from the Jackson Laboratory interleukin-1b (IL-1b) induced by bacteria or lipopolysaccharide (Bar Harbor, ME USA). LXRab+/+ and LXRab-/- mice (Sv129/ (LPS) (5, 6). Several mechanisms have been proposed to explain C57bl/6 background), were provided by David Mangelsdorf the anti-inflammatory actions of LXR ligands, including (University of Texas Southwestern, USA) and were maintained transrepression, or the induction of anti-inflammatory molecules. on standard chow under pathogen-free conditions. Mice aged 8- Importantly, Ito et al. elegantly described a mechanism underlying 12 weeks were used for experimental procedures following this antagonism, which appears to involve the LXR ligand-dependent Institutional Care Instructions (Bioethical Commission from induction of ABCA1 and the redistribution of membrane lipids Consejo Superior de Investigaciones Cientı́ficas). thereby resulting in reduced inflammatory signaling (7). Caveolin-1 protein (Cav-1) is the most common isoform Antibodies responsible for the formation of caveolae, a type of invaginated Antibodies against caveolin-1, flotillin-1, clathrin, total STAT3 lipid raft microdomains between 50-100 nm of the plasma and STAT1 and their phosphorylated forms were obtained from membrane enriched in cholesterol, which play an important BD Transduction Laboratories (Lexinton, KY). Monoclonal anti role in the regulation of various cellular functions including b−Actin was purchased from Sigma-Aldrich (St. Louis, MO) and endocytosis, transcytosis and cellular signaling (8). Cav-1 is also polyclonal anti-iNOS, anti-GFP and anti-COX2 were obtained a high affinity cholesterol-binding protein. In fact, the formation from Santa Cruz Biotechnology. ABCG1 Antibody (NB400-132) of caveolae and the expression of caveolin-1 are highly was from Novus Biologicals. ABCA1 and F4/80 were detected dependent on the availability of cholesterol (9, 10). Previous using specific anti serums, kindly provided by Michael L. studies have shown that these proteins participate in the Fitzgerald and Mason W. Freeman (MGH, Boston MA), and regulation of plasma lipoprotein metabolism, as well as Siamon Gordon (Oxford University), respectively. Alexa Fluor- cholesterol homeostasis, a process that must be adequately conjugated secondary antibodies were from Molecular Probes controlled to limit and avoid cholesterol accumulation and, (Eugene, OR). Horseradish peroxidase HRP- and gold- ultimately, prevent the development of atherosclerosis (11). In conjugated secondary antibodies were from Jackson this context, Cav-1 has been shown to participate in intracellular Inmmunoresearch Laboratories (West Grove, PA). trafficking of de novo synthesized cholesterol to the plasma membrane (12–15). Therefore, it is believed that the deficiency Cell Culture and Treatments of Cav-1 would lead to the accumulation of cholesterol in certain Macrophage cell line RAW 264.7 was obtained from the intracellular compartments (16, 17). Other studies have also American Type Culture Collection. RAW 264.7 cells were suggested that Cav-1 can participate in cholesterol efflux to cultured in Dulbecco’s modified Eagle’s medium (DMEM) extracellular acceptors (11, 18), however, very little is known containing 10% fetal bovine serum (FBS) 100 units/ml about the role of Cav-1 in cholesterol efflux induced by LXR- penicillin and streptomycin and 2mM L-glutamine at 37°C in ABCA1/G1 in macrophages. Despite that the presence of a humidified incubator at 5% CO2 95% O2. Culture reagents were caveolae-like invaginations in immune cells has been a purchased from BioWhittaker (Walkersville, MD). Mouse controversial topic (19), and that the major expression of Cav- peritoneal macrophages were isolated from mice by peritoneal 1 is primarily found in vascular endothelial cells, there is ample lavage using PBS 4 days after an intraperitoneal injection of evidence demonstrating that Cav-1 is also expressed and 1.5 ml of sterile thioglycollate broth (thioglycollate-elicited). Frontiers in Endocrinology | www.frontiersin.org 2 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages Macrophages were seeded in 35-mm dishes at 3x 106/well in Palo Alto, CA). The precipitated fraction was resuspended in RPMI 1640 medium supplemented with 10% FBS and penicillin/ lysis buffer and solubilized at 4°C. streptomycin (each at 100 units/ml). Unattached cells were washed off after 3 h with RPMI (twice), and the remaining Detergent-Free Caveolae Extraction macrophages were incubated overnight in RPMI supplemented Caveolae extraction from mouse peritoneal macrophages and with 10% FBS. On the following day, cells were washed twice Raw 264.7 cell cultures were carried out following the procedure again, incubated in 0.5% FBS RPMI, and treated as indicated. For described previously were TX-100 was replaced by sodium cell treatments we used the specific LXR agonists GW3965, carbonate and a sonication step was introduced to finely provided by Tim Willson and Jon Collins (GlaxoSmithKline). disrupt cellular membranes (33). Briefly, cells were T0901317 and the RXR agonist, LG268 obtained from TOCRIS. homogenized using a loose-fitting Dounce homogenizer (10 Ligands were dissolved in DMSO before use in cell culture. LXR strokes) and a sonicator (three 20-s bursts). 5mg of cellular ligands were used at 1 µM, whereas RXR ligand was used at 50 homogenate was then adjusted to 45% sucrose by the addition of nM. LPS from E. Coli was purchased from Sigma. 2 ml of 90% sucrose prepared in MBS (25 mM MES, pH 6.5, 150mM NaCl) and placed at the bottom of an ultracentrifuge Transfections tube. 5mg of cellular homogenate was transferred to a SW41-Ti The full length murine Caveolin-1 or GFP cDNA were cloned in- tube. A 5-35% discontinuous sucrose gradient was formed above frame in pCDNA3.1 vector (Invitrogen, Inc.). Twenty-four hours and centrifuged at 39,000 rpms for 20 h in an SW41 rotor before transfection, 2x106 RAW 264.7 cells were plated per 60- (Beckman Instruments, Palo Alto, CA) to obtain a total of 13 mm dish. On the day of transfection, 5 µg of plasmid DNA was fractions A light-scattering band confined to the 5–35% sucrose diluted in 200 µl of serum-free DMEM media. In a separate tube, interface was observed that contained caveolin but excluded 20 µl of Lipofectamine 2000 was diluted in 200 µl of serum-free most other cellular proteins. A volume of 20 µl form each DMEM media. The diluted DNA and Lipofectamine were then sucrose fraction obtained was analyzed by western blot with gently mixed and incubated at 25°C for 30 min. After the specific antibodies. incubation, 6 ml of serum-free RPMI media was added to the DNA/Lipofectamine mixture, mixed, and placed onto cells Protein Analysis by Western Blotting rinsed with serum-free DMEM media. The cells were and Bioplex ELISA incubated for 5 h at 37°C. Without washing, 3.6 ml of DMEM Equal amounts of each sample (25-50 µg) were electrophoresed media containing 20% FBS was added. The cells were grown for on sodium dodecyl sulfate–polyacrylamide gels electrophoresis 24 h. The media was removed, and DMEM media containing (SDS–PAGE) and transferred to nitrocellulose membranes. 10% FBS and 1.5 mg/ml Geneticin (G418) was added. Different Membranes were pre-incubated with 5% blotting grade blocker clones of antibiotic-resistant cells were isolated and tested for non-fat dry milk (Bio-Rad Laboratories, Hercules, CA, USA) in Cav-1 and GFP expression. Cells were grown under constant TBS with 0.1% Tween 20 (TBS-T) at room temperature for 1 h selection in medium containing 500 µg/ml Geneticin. and blotted over night at 4°C with the specific primary antibodies. Antibody-specific labeling was revealed by Total Protein Extracts and incubation with a HRP-conjugated goat anti-mouse or anti- Microsomal Purification rabbit secondary antibody (1:5000) and visualized with the For total protein extracts, cells were washed in PBS and scraped ECL chemiluminescence kit (Amersham Biosciences). Cytokine with lysis buffer (62.5 mM Tris-HCl, 1% SDS, 60 mM octyl- production in vitro by macrophages and its accumulation in the glucoside 10% glycerol, pH 6.8) containing protease inhibitors culture medium was quantified using an ELISA Bioplex kit (Bio- (Roche, Boehringer Mannheim). To obtain liver protein extracts, Rad) according to the manufacturer’s instructions. the tissue was briefly dissected and washed thoroughly with ice cold phosphate buffered saline and snap frozen in liquid RNA Isolation and Gene Expression nitrogen. Frozen tissue was mechanically homogenized in lysis RNA from liver and peritoneal macrophages were extracted using buffer with protease inhibitors. An aliquot of each extract Trizol reagent (Life Technologies, Inc). Levels proinflammatory was preserved for protein quantification by bicinchoninic acid genes like IL-6, IL-1b, iNOS, COX-2, and other mRNAs assay (32). Five per cent b-mercaptoethanol and 0.001% (caveolin-1 and ABCA1) were determined by quantitative reverse bromophenol blue were then added, and samples were boiled transcription real time (RT)-PCR (SYBRgreen) containing sense at 95°C for 5 min. For microsomal fractionation, cells plated in and antisense primer sequences of the tested genes as well as of three 100-mm wells were washed twice with ice-cold PBS 36B4 ribosomal protein (housekeeping gene) as we have previously and scraped down into PBS containing a protease inhibitor described (34). mixture. Cells were then sedimented and resuspended in 1 ml of hypotonic buffer (0.25 M sucrose, 20 mM Tricine, pH 7.8, Immunofluorescence Microscopy 1 mM EDTA) with protease inhibitors. After 20 min on ice, cells Peritoneal macrophages and Raw 264.7 cells were seeded on were disrupted using a Potter-Elvehjem homogenizer. Nuclei coverslips at a density of 0.5 x 106/well. For fluorescent labeling and cellular debris were removed by sedimenting the of lipid rafts, the cells were incubated with 2 µg/ml of CTxb homogenate at 1,000 g for 10 min at 4°C. The supernatant was Alexa 594 (Molecular Probes, Inc.) in 0.1% BSA-PBS for 20 min ultracentrifuged 1h at 100,000 g in TLA-100.1 rotor (Beckman, at 4°C prior to cell fixation. Cells were then fixed in 4% Frontiers in Endocrinology | www.frontiersin.org 3 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages paraformaldehyde (PFA) and permeabilized with 100% RESULTS methanol for 5 min at -20°C. For liver immunohistochemical analysis, tissues were dissected from the animal, embedded Caveolin-1 Expression Modulates in OCT compound (Tissue-Tek) and snap-frozen in liquid Inflammatory Responses and ABCA1 nitrogen and isopentane. 4 mm sections were air-dried, fixed Expression in RAW264.7 Cells with 4% PFA and permeabilized with 0.01% Triton X-100 in Previous studies have shown that ligand-activated LXRs down- PBS. The sections and the coverslips were then sequentially regulate the expression of inflammatory genes in macrophages incubated at room temperature in PBS containing 4% goat (6, 36–38), and other studies identified Cav-1 as an serum and 0.8% bovine serum albumin (BSA) for 60min, with immunomodulatory mediator in these cells (39, 40). In order to the indicated primary antibody over night at 4°C and with explore the possible implication of Cav-1 in the modulation LXR the fluorescence-tagged secondary antibodies in 0.8% BSA-PBS anti-inflammatory function, we analyzed the LPS-induced for 60 min. The coverslips were then mounted on glass slides cytokine production in various clones of RAW264.7 using Vectashield mountain medium fluorescence with DAPI macrophages expressing Cav-1 ectopically. RAW264.7 control (Vector Laboratories) and fluorescence signals were monitored cells (expressing green fluorescent protein, GFP) do not express using a Zeiss LSM 5 PASCAL Laser Scanning Microscope Cav-1 basally (Figure 1A). Based on the expression levels of Cav-1 (Carl Zeiss, Germany). in purified fractions of plasma membrane assessed by Western blot (Figure 1A), and the co-localization with GM-1 ganglioside, Electron-Microscopy Procedures a marker of lipid rafts labelled with a fluorescent choleric b- Culture cells were chemically fixed at 4°C with a mixture of subunit toxin, we chose RAW Cav-1 clone 1 for further analysis 2% PFA and 0.1% glutaraldehyde in PBS. After washing with (Figure 1B). Western blot and quantitative real-time PCR (qRT- PBS containing 50 mM glycine, cells were embedding in 12% PCR) experiments showed that LPS-dependent expression of gelatin and infused in 2.3 M sucrose. Mounted gelatin blocks iNOS and COX-2 (Figure 1C), as well as IL-6, IL-1b and were frozen in liquid nitrogen. Thin sections were prepared in MCP-1 (Figure 1D) was reduced in Cav-1 overexpressing cells an ultracryomicrotome (Leica EM Ultracut UC6/FC6, Vienna, compared with control GFP cells. Next, we explored whether Cav- Austria). Ultrathin cryosections were collected with 2% 1 expression could influence the expression of LXR target genes. methylcellulose in 2.3 M sucrose. Cryosections were incubated RNA expression of Abca1, Abcg1, Apoe and Srebf1 was similar in at room temperature on drops of 2% gelatine in PBS for 20 min RAW-GFP and RAW-Cav-1 cells (not shown). Since CAV-1 at 37°C, followed by 50 mM glycine in PBS during 15 min and function has been associated to cholesterol trafficking within the 10% FBS in PBS during 10 min and 5% FBS in PBS 5 min. Then plasma membrane, we explored whether expression of Cav-1 in they were incubated with a mouse anti-ABCA1 and rabbit anti- RAW cells influences the localization of the key protein involved caveolin 1 antibodies (1:50 both) in 5% FBS in PBS for 30 min. in cholesterol efflux ABCA1. Interestingly, we observed higher After three washes of PBS for 10 min, sections were incubated for levels of ABCA1 protein in whole cell lysates of Cav-1 20 min with anti-mouse coupled to 12 nm and anti-rabbit overexpressing macrophages compared to control cells (Figure coupled to 18 nm gold particles (Jackson ImmunoResearch, 1E), as well as in lipid raft fractionation experiments, were ABCA1 PA, USA). This was followed by three washes with drops of co-fractionated with Cav-1 and other markers like Flotillin-1 PBS for 10 min, two washes with distilled water. As a control (Flot-1) in rafts membranes (Figure 1F). Together, these results for non-specific binding of the colloidal gold-conjugated indicate that ectopic expression of Cav-1 in RAW264.7 cells antibody, the primary polyclonal antibody was omitted. The reduced inflammatory gene expression and promotes ABCA1 observations were done in an Electron Microscope Tecnai protein localization within membrane raft microdomains. Spirit (FEI Company, The Netherlands) with a CCD camera Altogether these results indicate that LXR and Cav-1 functions SIS Megaview III. exhibit a reciprocal influence on each other and suggest a possible cross-talk between LXR and Cav-1 signaling in macrophages. Cholesterol Efflux Assays Peritoneal macrophages from WT and Cav-1-/- mice were cultured at a density of 1 × 106 cells per well 1 day prior to Caveolin-1 Expression Is Reduced loading with 0.5 mCi/ml [3H]-cholesterol for 24 h with or in LXRab-/- Mouse Peritoneal without T0901317 (2 uM) for 12 h (35). Cells then were Macrophages and Liver washed twice with PBS and incubated in RPMI 1640 medium Since Cav-1 overexpression enhanced ABCA1 protein in lipid rafts supplemented with 2 mg/ml fatty acid-free bovine serum and promoted anti-inflammatory effects in macrophages, we albumin (FAFA media) in the presence of an Acetyl- assessed the abundance and distribution of Cav-1 in WT and Coenzyme A Acetyltransferase (ACAT) inhibitor (2 mM; LXRab-/- deficient mice. Our Western blot analysis in total cell Novartis Corporation, New York, NY, USA) for 4 h prior to lysates and in microsomal extracts from mouse peritoneal the addition of 50 ug/ml human ApoA1 in FAFA or HDL macrophages showed reduced Cav-1 expression in LXRab-/- cells (Intracell) media. Supernatants were collected after 6 h and compared to WT (Figure 2A), whereas no changes were observed expressed as a percentage of [3H]-cholesterol in the media per in other lipid rafts marker such as Flot-1. Additionally, Cav-1 total cell [ 3 H]-cholesterol content (total effluxed [ 3 H]- expression was also decreased in liver extracts from in LXRab-/- cholesterol + cell-associated [3H]-cholesterol). compared to WT mice (Figure 2B). Further, real-time qPCR Frontiers in Endocrinology | www.frontiersin.org 4 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages A B C D E F FIGURE 1 | Caveolin-1 expression modulates inflammatory genes and ABCA1 expression in RAW264.7 cells. (A) Western blot analysis of ectopically expressed Cav-1 in RAW264.7 cells (RAWCav-1) compared with RAW264.7 control cells overexpressing GFP (RAWGFP). a1-Na/K-ATPase protein was used to detect plasma membrane enrichment in microsomal preparations. Densitometric analysis of Cav-1 are shown below each blot and are referred to Total or MF in RAWCav-1 cells clone 1. (B) Colocalization of Cav-1 with lipid rafts in the plasma membrane stained with CTx-b in RAWCav-1cells. Scalebar: 10µm. (C) Representative Western blot analysis of iNOS and COX-2 in RAWCav-1 and RAWGFP cells. b-Actin was used as a loading control. Densitometric values of iNOS and COX-2 are shown below each blot. (D) qRT-PCR analysis of iNOS, IL-6, IL-1b and MCP-1 mRNA expression in RAWCav-1 and RAWGFP cells treated with 100ng/mL of LPS for 6 hours. Data are expressed as relative expression levels and correspond to the means ± SEM from three independent experiments performed in triplicate *P < 0.05 (significantly different from RAWGFP cells). (E) Representative Western blot analysis of ABCA1 in RAWCav-1 and RAWGFP cells in response to LXR/RXR ligands for 24h(GW3965 1µM+LG268 100nM). b-Actin was used as a loading control. Densitometric values of ABCA1 are shown below each blot. (F) Representative Western blot analysis of lipid raft fractionation in RAWCav-1 (left panel) and RAWGFP (right panel) cells showing the expression of Cav-1 and ABCA1 in response to LXR/RXR ligands for 24h (GW3965 1µM+LG268 100nM). Flotillin-1 was used as positive control of raft fractions and Clathrin as non-raft protein. analysis revealed a reduction of approximately 50% of Cav-1 mRNA Subcellular Distribution Caveolin-1 levels in both peritoneal macrophages and liver samples from and ABCA1 Is Controlled by LXR activity LXRab deficient mice (Figure 2C). Then, to better characterize Previous studies have reported contrasting data regarding the the localization of Cav-1 within the plasma membrane, we observed regulation of Cav-1 functions by cellular cholesterol in different the expression of the macrophage-specific membrane antigen F4/80 mouse or human macrophage cell models (41). However, given the and Cav-1 in WT and LXRab-/- macrophages. Confocal images well-known role for Cav-1 in cholesterol transport from showed double positive immunostaining of F4/80 and Cav-1 in WT endoplasmic reticulum to the plasma membrane and the recent peritoneal macrophages, while the colocalization of both proteins identification of the LXR targets, Gramd1/Aster, that control the was less evident in LXRab-/- macrophages due to reduced movement of cholesterol accessible pools within the plasma expression of Cav-1 in these cells (Figure 2D). Similar results membrane (42–44), we decided to explore whether LXR were found in Kupffer cells from liver sections of LXRab-/- mice activation would affect Cav-1 expression and subcellular compared to WT controls (Supplementary Figure 1). These results distribution in macrophages. To this end, we first performed indicate that LXR activity is important for the plasma membrane microscopy analysis to examine the patterns of Cav-1 localization of Cav-1 in macrophages. immunoreactivity within the plasma membrane during LXR Frontiers in Endocrinology | www.frontiersin.org 5 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages A B C D FIGURE 2 | Cav-1 expression is impaired in LXRab-/- mice. Representative Western blot analysis of Cav-1 in whole-cell lysates (Total) and microsomal fractions (MC) of peritoneal macrophages (A) and liver (B) from in WT and LXRab-/- mice. (C) mRNA expression of Cav-1 by real-time qRT-PCR in peritoneal macrophages and liver from WT and LXRab-/- mice. Data are expressed as relative expression levels and correspond to the means ± SEM from three independent experiments performed in triplicate *P < 0.05 (significantly different from WT [normalized to 1]). (D) Representative confocal images of Cav-1 expression (red) and F4/80 (green) in peritoneal macrophages from WT and LXRab-/- mice. Experiment was performed 3 independent times. Scalebar: 10µm. activation. Interestingly, cells treated with a combination of the expression and localization of ABCA1 and ABCG1 under the same synthetic LXR and RXR ligands (GW3965 and LG268; GW+LG) sucrose fractioning conditions. Western blot analysis showed that showed an intense staining of plasma membrane Cav-1 compared ABCA1 and ABCG1 proteins were mostly present in the lipid raft to the vehicle-treated cells (Figure 3A, left panel), while such fraction and it co-fractionated with Cav-1 in wild type increase was not observed in LXR-null macrophages (Figure 3A, macrophages but not in LXRab-/- macrophages (Figure 3B, both right panel). These results suggested a possible subcellular top and lower panels). Moreover, in agreement with role of Cav-1 relocalization of Cav-1 in response to LXR activation. To further as a cholesterol transport protein and the capacity of LXR to characterize the changes in Cav-1 localization in response to LXR control cholesterol pools within the cell, LXR activation promoted activation, we used a sodium carbonate detergent-free method to subcellular redistribution of cholesterol (stained with Filipin III), purify lipid rafts from peritoneal macrophages. Western blot together with Cav-1, in WT macrophages but it remained and analysis from sucrose gradient fractions using specific antibodies accumulated intracellularly in LXRab-/- cells (Supplementary against Cav-1, Flot-1, ABCA1 and ABCG1 showed that Cav-1 Figure 2). This may represent an abnormal localization of this protein expression appeared mainly at light buoyant fraction (F4- protein within the macrophage in cells lacking both isoforms of F5) containing the lipid raft membranes together with the raft LXR. Together, these results indicate that LXR activation promotes marker Flot-1, as well as in other intracellular organelles and ABCA1, ABCG1 and Cav-1 relocalization within the raft membranes in peritoneal macrophages (Figure 3B, upper panel). microdomains of the plasma membrane in murine macrophages. Interestingly, after LXR activation, Cav-1 expression was slightly increased in the raft fraction but decreased in the non-raft Cav-1 and ABCA1 Co-Localize in Mouse compartments, consistent with an intracellular redistribution of Peritoneal Macrophages Cav-1 dependent on LXR. Importantly, these effects of Cav-1 Despite of the well-established importance of ABCA1 in redistribution within raft fractions were abolished in LXRab-/- cholesterol transport and role of Cav-1 in the cholesterol macrophages (Figure 3B, lower panel). Next, we also assessed the homeostasis, the possible colocalization of these two proteins has Frontiers in Endocrinology | www.frontiersin.org 6 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages A B FIGURE 3 | Cav-1 cellular distribution is dependent on LXR. (A) Representative images showing the subcellular distribution of Cav-1 (green) by confocal microscopy in peritoneal macrophages from WT and LXRab-/- mice treated for 24 h with 1 mM GW3965 and 100 nM LG268 (G+L). Nuclei were stained with DAPI. Experiment was performed 3 independent times. Scalebar: 10µm. (B) Representative Western blot analysis of Cav-1 and ABCA1 and ABCG1 in lipid raft fractions from WT and LXRab-/- peritoneal macrophages treated for 24 h with G+L. not been fully identified at the ultrastructural level in macrophages. of their functionalities, with possible implications of Cav-1 Based on our previous results showing the co-fractionation of in LXR-dependent regulation of cholesterol metabolism ABCA1 with Cav-1 we decided to further characterize in depth the and inflammation. colocalization of both proteins in mouse macrophages. To do so, we performed electron microscopy studies of colloidal ABCA1 and The Anti-Inflammatory Actions Cav-1 gold particles. Individual immunostainings revealed that of LXR Agonists Are Decreased both particles were primarily located in the caveolae-like structures in Cav-1-/- Macrophages and intracellular cytoplasmic vesicles, as well as in other non- We and others have previously reported that cultured WT caveolae membranes (Supplementary Figure 3). We then macrophages with LXR agonists display reduced expression of performed combined labeling of ABCA1 and Cav-1 in WT and inflammatory markers in response to LPS. These anti- LXRab-/- macrophages. Figure 4 (upper micrographs) shows that inflammatory actions are abolished in macrophages from ABCA1 and Cav-1 were consistently found decorating the LXRab-/- mice (38, 45). In addition, given that overexpression of membrane of the same vesicles. Intriguingly, we were not able to Cav-1 in macrophages partially inhibited inflammatory cytokine find colocalization of immunogold particles of ABCA1 and Cav-1 production and LXR activation promoted Cav-1 redistribution in LXRab-/- macrophages Figure 4 (lower micrographs). These within the raft membrane domains, we hypothesized that Cav-1 results are in agreement with our previous co-fractionation of could be involved in LXR-dependent regulation of inflammation. Cav-1 and ABCA1 and suggest a possible reciprocal relationship To asses this, we compared the inflammatory outcomes of primary Frontiers in Endocrinology | www.frontiersin.org 7 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages FIGURE 4 | Colocalization of Cav-1 and ABCA1 within sub-cellular domains of peritoneal macrophages. Representative immuno-electron microscopy images showing Cav-1 and ABCA1 expression in peritoneal macrophages from WT and LXRab-/- mice. Scalebar: 200nm. peritoneal macrophages from wild type (WT) and Cav-1−/− mice and COX-2 expression by Western blotting (not shown). treated with LPS alone, or in combination with LXR/RXR ligands. Nevertheless, the ameliorated anti-inflammatory response of Interestingly, we found that the inhibitory capacity of LXR to LXR in the absence of Cav-1 was not due to differences in the block the production of pro-inflammatory cytokines, including expression of LXRa, LXRb or their target genes in Cav-1 deficient TNF-a and IL-1b, as well as other mediators such as IFN−g or cells (Figure 5B). These results suggest that Cav-1 expression is an GM-CSF was severely compromised in Cav-1-/- macrophages important element that facilitates full acquisition of LXR anti- (Figure 5A). Similar results were observed by analyzing iNOS inflammatory signaling in macrophages. Frontiers in Endocrinology | www.frontiersin.org 8 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages A B FIGURE 5 | Cav-1 influences anti-inflammatory effects of LXR in peritoneal macrophages. (A) Cytokine production in culture media of peritoneal macrophages from WT and Cav-1-/- mice pre-treated with 1 mM T0901317 and 1 mM 9-Cis Retinoic Acid for 18h hours prior to stimulation with LPS for another 24h. Data represent 3 independent experiments performed in triplicate *P < 0.05, **P < 0.01 (significantly different from WT in Ctrl conditions). (B) qRT-PCR analysis of LXRa, LXRb, ABCA1, ABCG1 and ApoE mRNA levels in peritoneal macrophages form WT and Cav-1-/- mice. Data are expressed as relative expression levels and correspond to the means ± SEM from three independent experiments performed in triplicate compared to WT and normalized to 1). Cholesterol Efflux to ApoAI and HDL Is macrophages (not shown). Our results show that activation of Impaired in Cav-1 Deficient Macrophages LXR induced the co-expression of ABCA1, ABCG1 and Cav-1 in The induction of ABCA1 and ABCG1 expression and activation of lipid raft microdomains within the plasma membrane. Since the the cholesterol efflux pathway (2, 3), together with inhibition of overall ABCA1 protein content was similar to WT in Cav-1-/- inflammation, are considered crucial steps for the atheroprotective macrophages, we reasoned whether lack of Cav-1 might have an functions of LXRs. Because our results suggest that ABCA1 and influence in ABCA1 distribution within the membrane. To Cav-1 proteins co-localize in cellular membranes and that this analyze this possibility, we cultured macrophages with LXR/RXR mutual cooperation is promoted by LXR activity, we decided to ligands and observed the localization of ABCA1 by confocal explore whether alteration of Cav-1 expression could influence microscopy. As shown in Supplementary Figure 4, LXR cholesterol efflux in macrophages. First, we assessed the expression activation induced a punctuate accumulation of ABCA1 protein of ABCA1 and ABCG1 in WT and Cav-1-/- macrophages. in the membrane of WT macrophages, whereas this distribution Although ABCG1 levels were slightly different in Cav-1 null was severely altered in Cav-1-/- macrophages. Taken together, this macrophages, induction of both ABC transporters was data indicate that Cav-1 participates in the distribution of ABCA1 appreciable in response to LXR agonists in WT and Cav-1 KO within the plasma membrane and in ApoAI-dependent macrophages (Figures 6A, C). Next, we assayed in vitro the cholesterol efflux in response to LXR activation in macrophages. cholesterol efflux to ApoAI and HDL in primary peritoneal macrophages from WT and Cav-1-deficient mice. As shown in Figures 6B, D, the cholesterol efflux to ApoAI and HDL was DISCUSSION significantly blunted in Cav-1-/- macrophages, indicating that Cav-1 expression is important for LXR-dependent cholesterol LXRs are transcription factors that regulate crucial processes efflux. Similar results were obtained in bone marrow derived in lipid metabolism and also exert important functions in Frontiers in Endocrinology | www.frontiersin.org 9 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages A B C D FIGURE 6 | Cav-1 influences subcellular distribution of ABCA1 and modulates ABCA1-dependent cholesterol efflux. (A) Western blot analysis of ABCA1 in peritoneal macrophages from WT and Cav-1-/- mice treated with or without or 1 mM T0901317 (T090) for 18 hours. Cav-1 was used to show its absence in the Cav-1-/- mice and HSP90 was used as a loading control. Experiment was performed at least 3 times. (B) Cholesterol efflux to apolipoprotein A1 (ApoA1) in peritoneal macrophages isolated from WT and Cav-1-/- mice stimulated with or without 3 mM of T090 for 16 hours. Data represent the mean ± SEM of triplicate samples (n = 3 per group; P < 0.05, (significantly different from WT in each treatment condition). (C) Western blot analysis of ABCG1 in peritoneal macrophages from WT and Cav-1-/- mice treated with or without or 1 mM T0901317 (T090) for 18 hours. Cav-1 was used to show its absence in the Cav-1-/- mice and HSP90 was used as a loading control. Experiment was performed at least 3 times. (D) Cholesterol efflux to HDL in peritoneal macrophages isolated from WT and Cav-1-/- mice stimulated with or without 3 mM of T090 for 16 hours. Data represent the mean ± SEM of triplicate samples (n = 3 per group; P < 0.05, (significantly different from WT in each treatment condition). *P < 0.05. inflammation and host immunity (46, 47). LXRs have emerged as Among caveolin proteins, Cav-1 is crucial for the formation of key regulators of whole body cholesterol homeostasis, in part caveolae and participates in several cellular processes, including through up-regulation of genes encoding plasma membrane endocytosis, cholesterol trafficking and signal transduction (52, 53). transporters ABCA1 and ABCG1 that facilitate cholesterol However, the role of Cav-1 during atherosclerosis is context and efflux from macrophages and other cell types (1, 2, 48). cell-type dependent. Hypercholesterolemic mice with complete loss Importantly, the same pathways that control cholesterol of Cav-1 or with Cav-1 targeting specifically in endothelial cells trafficking within the plasma membrane appeared as important demonstrated that Cav-1 function accelerates atherogenesis by regulators of inflammation and host defense (49, 50). In line with modulating endothelial lipoprotein transport and autophagy this, regulation of cholesterol efflux and the control of pathways (54, 55). In contrast, other studies using bone marrow inflammatory and antimicrobial responses in macrophages transplant of Cav-1-/- hematopoietic progenitors into LDLR-/- mice have been shown to be partially dependent on ABCA1 (7, 50, indicated that an important contribution of Cav-1 in macrophages 51). In the present study we provide an additional mechanistic is generally atheroprotective through modulation of macrophage clue that identifies Cav-1 as an important factor required for inflammation (56). Thus, these reports reconcile the apparent LXR-dependent functions in murine macrophages. Both the contrasting roles of Cav-1 in atherosclerosis and highlight the anti-inflammatory actions and cholesterol efflux to ApoAI or complexity and the importance of Cav-1 in lipid and inflammatory HDL promoted by LXR agonists are substantially diminished in pathways in the context of atherosclerosis. Also, all these studies the absence of Cav-1. Reciprocally, localization of Cav-1 within infer that LXRs and Cav-1 appear to participate in similar pathways lipid-raft membrane microdomains is impaired in LXR-deficient in macrophages, including phagocytosis, inflammation and macrophages. These findings reveal that a mutual cooperation cholesterol efflux. However, a deeper comprehension of how between Cav-1 and LXR pathways participates in macrophage LXR and Cav-1 pathways can affect each signaling reciprocally inflammatory and homeostatic responses. was not explored before. Frontiers in Endocrinology | www.frontiersin.org 10 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages The first experimental evidence that supported a mutual previous results by Parks and colleagues that showed increased crosstalk between Cav-1 and LXR came from our experiments membrane lipid raft content with reduced Cav-1 in raft fractions of forced Cav-1 expression in RAW264.7 macrophages that and hyper-inflammatory responses in ABCA1-deficient normally lack Cav-1. In this model, Cav-1 favors accumulation macrophages (63). Our studies demonstrate that LXR deficiency of ABCA1 mainly in lipid-raft domains in response to LXR in macrophages directs a reduction of Cav-1 within the lipid-raft ligands and a marked attenuation of inflammatory gene membrane domains that would promote delocalization of expression in response to LPS (Figure 1). Conversely, Cav-1 ABCA1 from raft/caveolar to non-raft membranes and other expression exhibited a reduction of ~50% in mRNA and total and subcellular locations. These molecular changes would probably microsomal protein expression in LXR-deficient macrophages influence the normal ABCA1 functions in macrophages. ABCA1 and liver, compared WT controls (Figure 2). Cav-1 down- activity is critical for cholesterol efflux and was also found to play regulation in LXR-null macrophages possibly suggests that LXR anti-inflammatory tasks in macrophages (7, 50, 63). Our data might be controlling Cav-1 expression through transcriptional indicate that some of the LXR-dependent functions that are regulation. However, our previously reported LXR ChIP-Seq and critically mediated by ABCA1, appear to rely on Cav-1 RNA profiling studies (57), have not demonstrated direct LXR expression. Using Cav-1 deficient macrophages, we demonstrate binding in the vicinity of the Cav-1 locus nor direct up-regulation that the ability of LXR ligands to control the expression of of Cav-1 expression in response to LXR ligands, indicating that inflammatory mediators depends greatly on Cav-1 expression. impaired Cav-1 expression in LXR-null macrophages could be These results are congruent with the studies by Ito et al. (7), which the result of an indirect regulation. Our observations indicate that clearly showed that induction of ABCA1 and subsequent ABCA1 and ABCG1 are concomitantly recruited with Cav-1 to uncoupling of TLR signaling from the plasma membrane lipid-raft microdomains (Figure 3). Our results are consistent mediate the anti-inflammatory actions of LXR. It seems with previous studies that showed a role for Cav-1 in ABCA1 and plausible from these studies and our results that Cav-1 and ABCG1 function in other cell types (58, 59). Thus, the occurrence ABCA1 participate in the regulation of inflammation by of Cav-1 in lipid-rafts of the plasma membrane augmented in controlling the recruitment of adaptor molecules that mediate response to LXR activation in macrophages and we hypothesize inflammation downstream of TLR. When the connection that the critical cholesterol efflux transporters ABCA1 and between ABCA1 and Cav-1 within lipid-rich microdomains is ABCG1 are important for this regulation. However, it is also enhanced by pre-treatment with LXR ligands, the magnitude of possible that the combined induction of several other direct LXR- inflammatory responses is attenuated. Furthermore, the regulated targets in response to elevated cholesterol levels assist in importance of Cav-1 expression in LXR-ABCA1 functions in Cav-1 recruitment to the plasma membrane in macrophages. macrophages has also implications in cholesterol efflux. Cav-1 Recently, elegant studies from Tontonoz and colleagues identified deficiency results in a marked reduction in LXR-dependent a family of proteins, called Aster, that facilitate the transport of cholesterol efflux to ApoA-I and HDL in macrophages. The excess accessible cholesterol in the plasma membrane to the defect in cholesterol efflux is not due to defect in ABCA1 or endoplasmic reticulum in response to LXR activation (42, 44). ABCG1 total protein expression, nor defects in LXRa or LXRb Thus, although the factors that are responsible for Cav-1 down- expression in Cav-1-/- macrophages. It is likely that the disruption regulation in LXR-deficient macrophages need further of caveolar microdomains by the loss of Cav-1 (64) affects the investigation, our observations indicate that an intact LXR membrane localization and function of ABCA1 and perhaps signaling is crucial for Cav-1 subcellular localization within the other proteins regulated by LXR involved in cholesterol efflux. plasma membrane in macrophages. Overall, our studies conclude that Cav-1 plays an important role Previous reports have described that overexpression of in LXR-mediated functions, both in inflammation and in ABCA1, ABCG1 and ABCG4 in HEK293 and CHO cells cholesterol efflux and that Cav-1 functions in macrophages influences cholesterol efflux through their activity in non-raft could provide additional intervention mechanisms to the LXR domains (60), based on experiments using detergents that transcriptional regulation of cholesterol efflux and inflammation. solubilize lipid membrane domains. Our experiments in primary macrophages, however, show that the majority of ABCA1 co-fractioned mainly with Cav-1 in light buoyant or DATA AVAILABILITY STATEMENT caveolar fractions. Furthermore, we demonstrate that ABCA1 and ABCG1 induction promoted by LXR activation takes place The raw data supporting the conclusions of this article will be mainly on caveolar fractions along with other lipid-rafts markers made available by the authors, without undue reservation. such as flotillin-1 (61). Our ultrastructural analyses (Figure 4) also indicate that ABCA1 and Cav-1 do not appear to interact physically but consistently co-localize within common membrane ETHICS STATEMENT microdomains. Importantly, our observations demonstrate that loss of LXR in macrophages, that greatly increases the The animal study was reviewed and approved by Comité Etica y intracellular cholesterol content (62), leads to reduced Cav-1 experimentació n animal Consejo Superior de Investigaciones and ABCA1 recruitment to lipid-rafts possibly by disrupting Cientı́ficas and Universidad Las Palmas de Gran Canaria, with caveolae microdomains. Our conclusions are in agreement with reference numbers PROEX171/2018 and OEBA-ULPGC 02/2015. Frontiers in Endocrinology | www.frontiersin.org 11 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages AUTHOR CONTRIBUTIONS Consorcio de Investigació n en Red de la Comunidad de Madrid, S2017/BMD-3686 and Fondo Europeo de Desarrollo Regional (to CMR and AC conceived the project. CMR, LB and AC LB). Ministerio de Ciencia, Investigació n y Universidades, and contributed to the conceptual design, figure preparation and Agencia Estatal de Investigació n Proyectos de I+D+i Retos acquired the funding for the project. CMR, MTP, VPM, MFF, Investigació n 2018 (RTI2018-095061-B-I00); TALENTO Grant APG, CT, JVR, PMR, MDS, UN, MCO, PGT and MC performed from Madrid Government, Spain (2017-T1/BMD-5333); experiments and data analysis. CMR and AC prepared draft Consejerı́a de Ciencia, Universidades e Innovació n Comunidad de manuscript. All authors revised the draft manuscript and Madrid, Spain (PEJD-2018-POST/BMD-8900 and PEDJ-2018-AI/ contributed to its editing. All authors contributed to the article BDM-9724) to CMR. and approved the submitted version. SUPPLEMENTARY MATERIAL ACKNOWLEDGMENTS The Supplementary Material for this article can be found online at: We are grateful to Dr. David Mangelsdorf (University of Texas https://www.frontiersin.org/articles/10.3389/fendo.2021.635923/ Southwestern, USA) for the LXR-deficient mice. We are indebted to full#supplementary-material Dr. Carlos Fernandez-Hernando (Yale University) for reagents and Supplementary Figure 1 | LXR deficiency in the liver results in decreased Cav-1 support, Drs. ML Fitzgerald and MW Freeman (MGH, Boston) for expression in Kupffer cells. Representative confocal images of Cav-1 expression the ABCA1 polyclonal antibody and Siamon Gordon (Oxford (red) and F4/80 (green) in livers from WT and LXRab-/- mice. Higher magnification of University) for F4/80 antibody. We also thank J. Collins (Glaxo the insets is shown in the right panels. Scalebar: 20µm. SmithKline) for the LXR and RXR ligands, and Drs. R. Mayoral- Supplementary Figure 2 | Cellular cholesterol redistribution upon LXR activation Moñibas, P. Martı́n-Sanz and MA del Pozo for early discussions. correlates with Cav-1 localization in peritoneal macrophages. Representative We thank MINECO FPI predoctoral fellowship granted to MCO images showing the subcellular distribution of Cav-1 (green) and Fillipin (blue) in (BES-2015-075339). Experimental work was supported by grants peritoneal macrophages isolated from WT and LXRab-/- mice and treated for 24 h from Ministerio de Ciencia, Investigació n y Universidades, y Fondo with 1 mM GW3965 and 100 nM LG268 (G+L). Scalebar: 10µm. Europeo de Desarrollo Regional (FEDER) Grant REF: PID2019- Supplementary Figure 3 | Cav-1 and ABCA1 localization in caveolae-like and 104284RB-I00/AEI/10.13039/501100011033 (to AC) and support intracellular vesicles. Representative electron microscopy images showing from Networks of Excellence from MINECO (Nuclear Receptors individual immunogold staining of Cav-1 and ABCA1 in peritoneal macrophages in Cancer, Metabolism and Inflammation [NuRCaMeIn] isolated from WT and LXRab-/- mice. Scalebar: 200nm. SAF2017-90604-REDT to AC. Ministerio de Economı́a, Industria Supplementary Figure 4 | Cav-1 influences subcellular distribution of ABCA1 y Competitividad, Ministerio de Ciencia, Investigació n y Representative images showing immunostaining of ABCA1 (red) by confocal Universidades, and Agencia Estatal de Investigació n (SAF2017- microscopy in peritoneal macrophages from WT and Cav-1-/- mice treated for 24 h 82436-R, RTC2017-6283-1), Centro de Investigació n Biomédica with 1 mM GW3965 and 100 nM LG268 (G+L). Nuclei were stained with DAPI. en Red en Enfermedades Cardiovasculares (CB16/11/00222), Experiment was performed 3 independent times. Scalebar: 20µm. 8. Boscher C, Nabi IR. Caveolin-1: Role in Cell Signaling. Adv Exp Med Biol REFERENCES (2012) 729:29–50. doi: 10.1007/978-1-4614-1222-9_3 1. Wang B, Tontonoz P. Liver X Receptors in Lipid Signalling and Membrane 9. Murata M, Peränen J, Schreiner R, Wieland F, Kurzchalia TV, Simons K. Homeostasis. Nat Rev Endocrinol (2018) 14:452–63. doi: 10.1038/s41574-018- VIP21/Caveolin is a Cholesterol-Binding Protein. Proc Natl Acad Sci USA 0037-x (1995) 92:10339–43. doi: 10.1073/pnas.92.22.10339 2. Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, et al. 10. Frank PG, Marcel YL, Connelly MA, Lublin DM, Franklin V, Williams DL, Control of Cellular Cholesterol Efflux by the Nuclear Oxysterol Receptor LXRa. et al. Stabilization of Caveolin-1 by Cellular Cholesterol and Scavenger Proc Natl Acad Sci USA (2000) 97:12097–102. doi: 10.1073/pnas.200367697 Receptor Class B Type I. Biochemistry (2002) 41:11931–40. doi: 10.1021/ 3. Kennedy MA, Barrera GC, Nakamura K, Baldá n Á , Tarr P, Fishbein MC, et al. bi0257078 ABCG1 has a Critical Role in Mediating Cholesterol Efflux to HDL and 11. Frank PG, Galbiati F, Volonte D, Razani B, Cohen DE, Marcel YL, et al. Preventing Cellular Lipid Accumulation. Cell Metab (2005) 1:121–31. Influence of Caveolin-1 on Cellular Cholesterol Efflux Mediated by High- doi: 10.1016/j.cmet.2005.01.002 Density Lipoproteins. Am J Physiol - Cell Physiol (2001) 280:1204–14. 4. Joseph SB, McKilligin E, Pei L, Watson MA, Collins AR, Laffitte BA, et al. doi: 10.1152/ajpcell.2001.280.5.c1204 Synthetic LXR Ligand Inhibits the Development of Atherosclerosis in Mice. 12. Pol A, Luetterforst R, Lindsay M, Heino S, Ikonen E, Parton RG. A Caveolin Proc Natl Acad Sci USA (2002) 99:7604–9. doi: 10.1073/pnas.112059299 Dominant Negative Mutant Associates With Lipid Bodies and Induces 5. Joseph SB, Bradley MN, Castrillo A, Bruhn KW, Mak PA, Pei L, et al. LXR- Intracellular Cholesterol Imbalance. J Cell Biol (2001) 152:1057–70. Dependent Gene Expression Is Important for Macrophage Survival and the doi: 10.1083/jcb.152.5.1057 Innate Immune Response. Cell (2004) 119:299–309. doi: 10.1016/j.cell.2004.09.032 13. Fielding CJ, Fielding PE. Membrane Cholesterol and the Regulation of Signal 6. Castrillo A, Tontonoz P. Nuclear Receptors in Macrophage Biology: At the Transduction. Biochem Soc Trans (2004) 32:65–9. doi: 10.1042/BST0320065 Crossroads of Lipid Metabolism and Inflammation. Annu Rev Cell Dev Biol 14. Fielding CJ, Fielding PE. Relationship Between Cholesterol Trafficking and (2004) 20:455–80. doi: 10.1146/annurev.cellbio.20.012103.134432 Signaling in Rafts and Caveolae. Biochim Biophys Acta - Biomembr (2003) 7. Ito A, Hong C, Rong X, Zhu X, Tarling EJ, Hedde PN, et al. Lxrs Link 1610:219–28. doi: 10.1016/S0005-2736(03)00020-8 Metabolism to Inflammation Through Abca1-Dependent Regulation of 15. Fielding CJ, Bist A, Fielding PE. Intracellular Cholesterol Transport in Membrane Composition and TLR Signaling. Elife (2015) 4:e08009. Synchronized Human Skin Fibroblasts. Biochemistry (1999) 38:2506–13. doi: 10.7554/eLife.08009 doi: 10.1021/bi981012o Frontiers in Endocrinology | www.frontiersin.org 12 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages 16. Ostermeyer AG, Paci JM, Zeng Y, Lublin DM, Munro S, Brown DA. Receptor Pathway. Mol Cell Biol (2019) 39(22):e00170–19. doi: 10.1128/ Accumulation of Caveolin in the Endoplasmic Reticulum Redirects the mcb.00170-19 Protein to Lipid Storage Droplets. J Cell Biol (2001) 152:1071–8. 35. Ramírez CM, Rotllan N, Vlassov AV, Dávalos A, Li M, Goedeke L, et al. doi: 10.1083/jcb.152.5.1071 Control of Cholesterol Metabolism and Plasma High-Density Lipoprotein 17. Bosch M, Marı́ M, Herms A, Ferná ndez A, Fajardo A, Kassan A, et al. Levels by microRNA-144. Circ Res (2013) 112:1592–601. doi: 10.1161/ Caveolin-1 Deficiency Causes Cholesterol-Dependent Mitochondrial CIRCRESAHA.112.300626 Dysfunction and Apoptotic Susceptibility. Curr Biol (2011) 21:681–6. 36. Tabraue C, Lara PC, De Mirecki-Garrido M, De La Rosa JV, Ló pez-Blanco F, doi: 10.1016/j.cub.2011.03.030 Ferná ndez-Pé rez L, et al. LXR Signaling Regulates Macrophage Survival and 18. Luo DX, Cao DL, Xiong Y, Peng XH, Liao DF. A Novel Model of Cholesterol Inflammation in Response to Ionizing Radiation. Int J Radiat Oncol Biol Phys Efflux From Lipid-Loaded Cells. Acta Pharmacol Sin (2010) 31:1243–57. (2019) 104:913–23. doi: 10.1016/j.ijrobp.2019.03.028 doi: 10.1038/aps.2010.93 37. Michael DR, Ashlin TG, Buckley ML, Ramji DP. Liver X Receptors, 19. Fiala GJ, Minguet S. Caveolin-1: The Unnoticed Player in TCR and BCR Atherosclerosis and Inflammation. Curr Atheroscler Rep (2012) 14:284–93. Signaling. Advances in Immunology (2018) 137:83–133. doi: 10.1016/ doi: 10.1007/s11883-012-0239-y bs.ai.2017.12.002 38. Castrillo A, Joseph SB, Marathe C, Mangelsdorf DJ, Tontonoz P. Liver X 20. Sowa G. Role of Caveolin Proteins in Sepsis. Pediatr Ther (2013) 03:S2-001. Receptor-Dependent Repression of Matrix Metalloproteinase-9 Expression in doi: 10.4172/2161-0665.s2-001 Macrophages. J Biol Chem (2003) 278:10443–9. doi: 10.1074/jbc.M213071200 21. Jiao H, Zhang Y, Yan Z, Wang Z-G, Liu G, Minshall RD, et al. Caveolin-1 Tyr 39. Wang XM, Kim HP, Song R, Choi AMK. Caveolin-1 Confers 14 Phosphorylation Induces Interaction With TLR4 in Endothelial Cells and Antiinflammatory Effects in Murine Macrophages Via the MKK3/p38 Mediates MyD88-Dependent Signaling and Sepsis-Induced Lung MAPK Pathway. Am J Respir Cell Mol Biol (2006) 34:434–42. doi: 10.1165/ Inflammation. J Immunol (2013) 191:6191–9. doi: 10.4049/jimmunol.1300873 rcmb.2005-0376OC 22. Savio LEB, De Andrade Mello P, Santos SACS, De Sousa JC, Oliveira SDS, 40. Del Favero G, Mayer RM, Dellafiora L, Janker L, Niederstaetter L, Dall’Asta C, Minshall RD, et al. P2X7 Receptor Activation Increases Expression of et al. Structural Similarity With Cholesterol Reveals Crucial Insights Into Caveolin-1 and Formation of Macrophage Lipid Rafts, Thereby Boosting Mechanisms Sustaining the Immunomodulatory Activity of the Mycotoxin CD39 Activity. J Cell Sci (2020) 133:jcs237560. doi: 10.1242/JCS.237560 Alternariol. Cells (2020) 9:847. doi: 10.3390/cells9040847 23. Takamura N, Yamaguchi Y, Watanabe Y, Asami M, Komitsu N, Aihara M. 41. Gargalovic P, Dory L. Caveolin-1 and Caveolin-2 Expression in Mouse Downregulated Caveolin-1 Expression in Circulating Monocytes may Macrophages: High Density Lipoprotein 3-Stimulated Secretion and a Lack Contribute to the Pathogenesis of Psoriasis. Sci Rep (2019) 9:125. of Significant Subcellular Co-Localization. J Biol Chem (2001) 276:26164–70. doi: 10.1038/s41598-018-36767-5 doi: 10.1074/jbc.M011291200 24. Codrici E, Albulescu L, Popescu ID, Mihai S, Enciu AM, Albulescu R, et al. 42. Ferrari A, He C, Kennelly JP, Sandhu J, Xiao X, Chi X, et al. Aster Proteins Caveolin-1-Knockout Mouse as a Model of Inflammatory Diseases. J Immunol Regulate the Accessible Cholesterol Pool in the Plasma Membrane. Mol Cell Res (2018) 2018:10. doi: 10.1155/2018/2498576 Biol (2020) 40:e00255-20. doi: 10.1128/mcb.00255-20 25. Herek TA, Robinson JE, Heavican TB, Amador C, Iqbal J, Cutucache CE. 43. Naito T, Ercan B, Krshnan L, Triebl A, Koh DHZ, Wei FY, et al. Movement of Caveolin-1 is Dispensable for Early Lymphoid Development, But Plays a Role Accessible Plasma Membrane Cholesterol by GRAMD1 Lipid Transfer in the Maintenance of the Mature Splenic Microenvironment. BMC Res Notes Protein Complex. Elife (2019) 8:e51401. doi: 10.7554/eLife.51401 (2018) 11:470. doi: 10.1186/s13104-018-3583-3 44. Sandhu J, Li S, Fairall L, Pfisterer SG, Gurnett JE, Xiao X, et al. Aster Proteins 26. Oyarce C, Cruz-Gomez S, Galvez-Cancino F, Vargas P, Moreau HD, Diaz- Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Valdivia N, et al. Caveolin-1 Expression Increases Upon Maturation in Mammalian Cells. Cell (2018) 175:514–29.e20. doi: 10.1016/j.cell.2018.08.033 Dendritic Cells and Promotes Their Migration to Lymph Nodes Thereby 45. Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P. Reciprocal Favoring the Induction of CD8+ T Cell Responses. Front Immunol (2017) Regulation of Inflammation and Lipid Metabolism by Liver X Receptors. Nat 8:1794. doi: 10.3389/fimmu.2017.01794 Med (2003) 9:213–9. doi: 10.1038/nm820 27. Frank PG, Cheung MWC, Pavlides S, Llaverias G, Park DS, Lisanti MP. 46. Schulman IG. Liver X Receptors Link Lipid Metabolism and Inflammation. Caveolin-1 and Regulation of Cellular Cholesterol Homeostasis. Am J Physiol FEBS Lett (2017) 591:2978–91. doi: 10.1002/1873-3468.12702 - Hear Circ Physiol (2006) 291:H677–86. doi: 10.1152/ajpheart.01092.2005 47. Gları́a E, Letelier NA, Valledor AF. Integrating the Roles of Liver X Receptors 28. Gargalovic P, Dory L. Cellular Apoptosis is Associated With Increased in Inflammation and Infection: Mechanisms and Outcomes. Curr Opin Caveolin-1 Expression in Macrophages. J Lipid Res (2003) 44:1622–32. Pharmacol (2020) 53:55–65. doi: 10.1016/j.coph.2020.05.001 doi: 10.1194/jlr.M300140-JLR200 48. Lesprit E, Escudier E, Roger G, Prulière V, Lenoir G, Reinert P, et al. 29. Qin L, Zhu N, Ao B-X, Liu C, Shi Y-N, Du K, et al. Caveolae and Caveolin-1 Characterization of Inflammatory Reaction in Upper Airways of Cystic Integrate Reverse Cholesterol Transport and Inflammation in Atherosclerosis. Fibrosis Patients. Histol Histopathol (2000) 15:395–402. doi: 10.14670/HH- Int J Mol Sci (2016) 17:429. doi: 10.3390/ijms17030429 15.395 30. Lei MG, Tan X, Qureshi N, Morrison DC. Regulation of Cellular Caveolin-1 49. Kidani Y, Bensinger SJ. Liver X Receptor and Peroxisome Proliferator- Protein Expression in Murine Macrophages by Microbial Products. Infect Activated Receptor as Integrators of Lipid Homeostasis and Immunity. Immun (2005) 73:8136–43. doi: 10.1128/IAI.73.12.8136-8143.2005 Immunol Rev (2012) 249:72–83. doi: 10.1111/j.1600-065X.2012.01153.x 31. Tsai TH, Tam K, Chen SF, Liou JY, Tsai YC, Lee YM, et al. Deletion of 50. Zhu X, Lee JY, Timmins JM, Brown JM, Boudyguina E, Mulya A, et al. Caveolin-1 Attenuates LPS/GalN-Induced Acute Liver Injury in Mice. J Cell Increased Cellular Free Cholesterol in Macrophage-Specific Abca1 Knock- Mol Med (2018) 22:5573–82. doi: 10.1111/jcmm.13831 Out Mice Enhances Pro-Inflammatory Response of Macrophages. J Biol Chem 32. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano (2008) 283:22930–41. doi: 10.1074/jbc.M801408200 MD, et al. Measurement of Protein Using Bicinchoninic Acid. Anal Biochem 51. Iio A, Ohguchi K, Iinuma M, Nozawa Y, Ito M. Hesperetin Upregulates (1985) 150:76–85. doi: 10.1016/0003-2697(85)90442-7 ABCA1 Expression and Promotes Cholesterol Efflux From THP-1 33. Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP. Co- Macrophages. J Nat Prod (2012) 75:563–6. doi: 10.1021/np200696r Purification and Direct Interaction of Ras With Caveolin, an Integral 52. Harris J, Werling D, Hope JC, Taylor G, Howard CJ. Caveolae and Caveolin in Membrane Protein of Caveolae Microdomains: Detergent-Free Purification Immune Cells: Distribution and Functions. Trends Immunol (2002) 23:158– of Caveolae Membranes. J Biol Chem (1996) 271:9690–7. doi: 10.1074/ 64. doi: 10.1016/S1471-4906(01)02161-5 jbc.271.16.9690 53. Ni K, Wang C, Carnino JM, Jin Y. The Evolving Role of Caveolin-1: A Critical 34. Ferná ndez-de Frutos M, Galá n-Chilet I, Goedeke L, Kim B, Pardo-Marqué s V, Regulator of Extracellular Vesicles. Med Sci (Basel Switzerland) (2020) 8:46. Pé rez-Garcı́a A, et al. MicroRNA 7 Impairs Insulin Signaling and Regulates doi: 10.3390/medsci8040046 Ab Levels through Posttranscriptional Regulation of the Insulin Receptor 54. Zhang X, Ramı́rez CM, Aryal B, Madrigal-Matute J, Liu X, Diaz A, et al. Cav-1 Substrate 2, Insulin Receptor, Insulin-Degrading Enzyme, and Liver X (Caveolin-1) Deficiency Increases Autophagy in the Endothelium and Frontiers in Endocrinology | www.frontiersin.org 13 May 2021 | Volume 12 | Article 635923 Ramı´rez et al. LXR/Cav-1 Signaling in Macrophages Attenuates Vascular Inflammation and Atherosclerosis. Arterioscler Thromb Phagocytosis in Tangier Cells. Mol Biol Cell (2004) 15:5399–407. Vasc Biol (2020) 1:1510–22. doi: 10.1161/ATVBAHA.120.314291 doi: 10.1091/mbc.E04-03-0182 55. Ramı́rez CM, Zhang X, Bandyopadhyay C, Rotllan N, Sugiyama MG, Aryal B, 62. Lee SD, Tontonoz P. Liver X Receptors at the Intersection of Lipid et al. Caveolin-1 Regulates Atherogenesis by Attenuating Low-Density Metabolism and Atherogenesis. Atherosclerosis (2015) 242:29–36. Lipoprotein Transcytosis and Vascular Inflammation Independently of doi: 10.1016/j.atherosclerosis.2015.06.042 Endothelial Nitric Oxide Synthase Activation. Circulation (2019) 140:225– 63. Zhu X, Owen JS, Wilson MD, Li H, Griffiths GL, Thomas MJ, et al. 39. doi: 10.1161/CIRCULATIONAHA.118.038571 Macrophage ABCA1 Reduces MyD88-Dependent Toll-Like Receptor 56. Pavlides S, Gutierrez-Pajares JL, Katiyar S, Jasmin JF, Mercier I, Walters R, Trafficking to Lipid Rafts by Reduction of Lipid Raft Cholesterol. J Lipid et al. Caveolin-1 Regulates the Anti-Atherogenic Properties of Macrophages. Res (2010) 51:3196–206. doi: 10.1194/jlr.M006486 Cell Tissue Res (2014) 358:821–31. doi: 10.1007/s00441-014-2008-4 64. Li J, Scherl A, Medina F, Frank PG, Kitsis RN, Tanowitz HB, et al. Impaired 57. Ramó n-Vá zquez A, de la Rosa JV, Tabraue C, Lopez F, Dı́az-Chico BN, Bosca Phagocytosis in Caveolin-1 Deficient Macrophages. Cell Cycle (2005) 4:1599– L, et al. Common and Differential Transcriptional Actions of Nuclear 607. doi: 10.4161/cc.4.11.2117 Receptors Liver X Receptors a and b in Macrophages. Mol Cell Biol (2019) 39:e00376-18. doi: 10.1128/mcb.00376-18 Conflict of Interest: The authors declare that the research was conducted in the 58. Gu HM, Wang FQ, Zhang DW. Caveolin-1 Interacts With ATP Binding absence of any commercial or financial relationships that could be construed as a Cassette Transporter G1 (ABCG1) and Regulates ABCG1-Mediated potential conflict of interest. Cholesterol Efflux. Biochim Biophys Acta - Mol Cell Biol Lipids (2014) 1841:847–58. doi: 10.1016/j.bbalip.2014.02.002 The handling editor declared a past co-authorship with one of the authors AC. 59. Wang F, Gu H, Zhang D. Caveolin-1 and ATP Binding Cassette Transporter A1 and G1-Mediated Cholesterol Efflux. Cardiovasc Hematol Disord Targets Copyright © 2021 Ramı ́rez, Torrecilla-Parra, Pardo-Marqués, de-Frutos, Pérez- (2014) 14:142–8. doi: 10.2174/1871529x14666140505122802 Garcı ́a, Tabraue, de la Rosa, Martı ́n-Rodriguez, Dı ́az-Sarmiento, Nuñez, Orizaola, 60. Sano O, Ito S, Kato R, Shimizu Y, Kobayashi A, Kimura Y, et al. Abca1, Trave ́s, Camps, Boscá and Castrillo. This is an open-access article distributed under ABCG1, and ABCG4 Are Distributed to Distinct Membrane Meso-Domains the terms of the Creative Commons Attribution License (CC BY). The use, distribution and Disturb Detergent-Resistant Domains on the Plasma Membrane. PloS or reproduction in other forums is permitted, provided the original author(s) and the One (2014) 9:e109886. doi: 10.1371/journal.pone.0109886 copyright owner(s) are credited and that the original publication in this journal is 61. Bared SM, Buechler C, Boettcher A, Dayoub R, Sigruener A, Grandl M, et al. cited, in accordance with accepted academic practice. No use, distribution or Association of ABCA1 With Syntaxin 13 and Flotillin-1 and Enhanced reproduction is permitted which does not comply with these terms. Frontiers in Endocrinology | www.frontiersin.org 14 May 2021 | Volume 12 | Article 635923