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Cited by 59Atherosclerosis and Cardiovascular DiseasesCholesterol and Lipid MetabolismSphingolipid Metabolism and Signaling

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Co-Stimulation of PAFR and CD36 Is Required for oxLDL-Induced Human Macrophages Activation

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Rios, Francisco J. O., Koga, Mariana M., Ferracini, Matheus, Jancar, Sonia·2012·PLoS ONE
DOI 10.1371/journal.pone.0036632PMC PMC3343035
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Abstract

The oxidative process of LDL particles generates molecules which are structurally similar to platelet-activating factor (PAF), and some effects of oxidized LDL (oxLDL) have been shown to be dependent on PAF receptor (PAFR) activation. In a previous study, we showed that PAFR is required for upregulation of CD36 and oxLDL uptake. In the present study we analyzed the molecular mechanisms activated by oxLDL in human macrophages and the contribution of PAFR to this response. Human adherent monocytes/macrophages were stimulated with oxLDL. Uptake of oxLDL and CD36 expression were determined by flow cytometry; MAP kinases and Akt phosphorylation by Western blot; IL-8 and MCP-1 concentration by ELISA and mRNA expression by real-time PCR. To investigate the participation of the PI3K/Akt pathway, Gαi-coupled protein or PAFR, macrophages were treated with LY294002, pertussis toxin or with the PAFR antagonists WEB2170 and CV3988, respectively before addition of oxLDL. It was found that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the MAPK (p38 and JNK). Phosphorylation of Akt requires the engagement of PAFR and a Gαi-coupled protein. The upregulation of CD36 protein and the uptake of oxLDL as well as the IL-8 production are dependent on PI3K/Akt pathway activation. The increased CD36 protein expression is dependent on PAFR and Gαi-coupled protein. Transfection studies using HEK 293t cells showed that oxLDL uptake occurs with either PAFR or CD36, but IL-8 production requires the co-transfection of both PAFR and CD36. These findings show that PAFR has a pivotal role in macrophages response to oxLDL and suggest that pharmacological intervention at the level of PAFR activation might be beneficial in atherosclerosis.

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Figure 1 oxLDL activates the PI3k/Akt pathway in a PAFR and Gαi coupled protein-dependent manner. Adherent monocytes/macrophages were treated with WEB2170 (50 µM, 30 min) (A) or PTX (600 ng/mL, 18 h) (B) and stimulated with oxLDL (30 µg/mL) for 10 min. Western blot analysis of cell lysates were performed using antibodies to phosphorylated and non-phosphorylated forms of Akt. Data are presented as mean ± SEM of four donors. Protein expression was quantified by the AlphaEaseFC™ software V3.2 beta (Alpha Innotech). The autoradiographs show one representative experiment. * p<0.05 comparing oxLDL-stimulated with the non-stimulated cells (cont). # p<0.05 comparing cells treated with WEB2170 or PTX with non-treated cells.
Figure 1 oxLDL activates the PI3k/Akt pathway in a PAFR and Gαi coupled protein-dependent manner. Adherent monocytes/macrophages were treated with WEB2170 (50 µM, 30 min) (A) or PTX (600 ng/mL, 18 h) (B) and stimulated with oxLDL (30 µg/mL) for 10 min. Western blot analysis of cell lysates were performed using antibodies to phosphorylated and non-phosphorylated forms of Akt. Data are presented as mean ± SEM of four donors. Protein expression was quantified by the AlphaEaseFC™ software V3.2 beta (Alpha Innotech). The autoradiographs show one representative experiment. * p<0.05 comparing oxLDL-stimulated with the non-stimulated cells (cont). # p<0.05 comparing cells treated with WEB2170 or PTX with non-treated cells.CC BY · PubMed Central
Figure 2 PI3k/Akt pathway is required for p38 and JNK MAPK activation induced by oxLDL. Adherent monocytes/macrophages were treated with LY294002 (10 µM, 30 min) and then stimulated with oxLDL (30 µg/mL) for 10 min. The MAPK Western blot analysis of cell lysates were performed using antibodies to phosphorylated and non-phosphorylated forms of ERK1/2, p38 and JNK (A, B and C, respectively). The autoradiographs show one representative experiment. Graph data are presented as mean ± SEM of four donors. Protein expression was quantified by the AlphaEaseFC™ software V3.2 beta (Alpha Innotech). * p<0.05 comparing oxLDL-stimulated with the non-stimulated cells (cont). # p<0.05 comparing cells treated with LY294002 with non-treated cells.
Figure 2 PI3k/Akt pathway is required for p38 and JNK MAPK activation induced by oxLDL. Adherent monocytes/macrophages were treated with LY294002 (10 µM, 30 min) and then stimulated with oxLDL (30 µg/mL) for 10 min. The MAPK Western blot analysis of cell lysates were performed using antibodies to phosphorylated and non-phosphorylated forms of ERK1/2, p38 and JNK (A, B and C, respectively). The autoradiographs show one representative experiment. Graph data are presented as mean ± SEM of four donors. Protein expression was quantified by the AlphaEaseFC™ software V3.2 beta (Alpha Innotech). * p<0.05 comparing oxLDL-stimulated with the non-stimulated cells (cont). # p<0.05 comparing cells treated with LY294002 with non-treated cells.CC BY · PubMed Central
Figure 3 PI3K/Akt pathway is required for oxLDL induced upregulation of CD36 expression (A) oxLDL uptake (B) and IL-8 production (C). Adherent human monocytes/macrophages were treated with LY294002 (10 µM, 30 min), and then stimulated with oxLDL (30 µg/mL) for 24 h. The expression of CD36 was measured by FACS and IL-8 production by ELISA. For the uptake evaluation, stimulated cells were washed and incubated with FITC-oxLDL for 1 h and uptake was measured by FACS. Data are presented as mean ± SEM of the mean fluorescence Intensity of five donors. * p<0.05 comparing oxLDL stimulated with non-stimulated cells (cont). # p<0.05 comparing cells treated with LY294002 with non-treated cells.
Figure 3 PI3K/Akt pathway is required for oxLDL induced upregulation of CD36 expression (A) oxLDL uptake (B) and IL-8 production (C). Adherent human monocytes/macrophages were treated with LY294002 (10 µM, 30 min), and then stimulated with oxLDL (30 µg/mL) for 24 h. The expression of CD36 was measured by FACS and IL-8 production by ELISA. For the uptake evaluation, stimulated cells were washed and incubated with FITC-oxLDL for 1 h and uptake was measured by FACS. Data are presented as mean ± SEM of the mean fluorescence Intensity of five donors. * p<0.05 comparing oxLDL stimulated with non-stimulated cells (cont). # p<0.05 comparing cells treated with LY294002 with non-treated cells.CC BY · PubMed Central
Figure 4 PAFR and Gαi-coupled protein is required for upregulation of CD36 protein and mRNA expression. Adherent human monocytes/macrophages were treated with WEB2170 (50 µM, 30 min) or with pertussis toxin (PTX - 600 ng/mL, 18 h) and then stimulated with oxLDL (30 µg/mL) or PAF (10−6 M). The expression of CD36 was measured by FACS after 24 h (A, C and D) and the mRNA levels after 5 h (B). Data are presented as mean ± SEM of the mean fluorescence intensity of five donors (A–B) or one representative experiment (C–D). * p<0.05 comparing oxLDL stimulated with non-stimulated cells (cont). # p<0.05 comparing cells treated with PTX, WEB2170 or CV3988, with non-treated cells.
Figure 4 PAFR and Gαi-coupled protein is required for upregulation of CD36 protein and mRNA expression. Adherent human monocytes/macrophages were treated with WEB2170 (50 µM, 30 min) or with pertussis toxin (PTX - 600 ng/mL, 18 h) and then stimulated with oxLDL (30 µg/mL) or PAF (10−6 M). The expression of CD36 was measured by FACS after 24 h (A, C and D) and the mRNA levels after 5 h (B). Data are presented as mean ± SEM of the mean fluorescence intensity of five donors (A–B) or one representative experiment (C–D). * p<0.05 comparing oxLDL stimulated with non-stimulated cells (cont). # p<0.05 comparing cells treated with PTX, WEB2170 or CV3988, with non-treated cells.CC BY · PubMed Central
Figure 5 oxLDL induces IL-8 and MCP-1 in a PAFR-dependent manner. Adherent human monocytes/macrophages were treated with WEB2170 (50 µM) or CV3988 (10 µM) for 30 min and stimulated with oxLDL (30 µg/mL). The mRNA levels for MCP-1 and IL-8 were evaluated after 5 h (n = 4) (A). The IL-8 concentration was measured in the supernatants after 24 h (n = 3) (B). THP-1 cells stimulated with different concentrations of oxLDL or LDL, the IL-8 concentration was measured after 24 h (C). Data are presented as mean ± SEM. * p<0.05 comparing oxLDL stimulated with non-stimulated cells (cont). # p<0.05 comparing cells treated with WEB2170 or CV3988 with non-treated cells.
Figure 5 oxLDL induces IL-8 and MCP-1 in a PAFR-dependent manner. Adherent human monocytes/macrophages were treated with WEB2170 (50 µM) or CV3988 (10 µM) for 30 min and stimulated with oxLDL (30 µg/mL). The mRNA levels for MCP-1 and IL-8 were evaluated after 5 h (n = 4) (A). The IL-8 concentration was measured in the supernatants after 24 h (n = 3) (B). THP-1 cells stimulated with different concentrations of oxLDL or LDL, the IL-8 concentration was measured after 24 h (C). Data are presented as mean ± SEM. * p<0.05 comparing oxLDL stimulated with non-stimulated cells (cont). # p<0.05 comparing cells treated with WEB2170 or CV3988 with non-treated cells.CC BY · PubMed Central
Figure 6 CD36 and PAFR cooperatively mediate FITC-oxLDL uptake and IL-8 production. HEK 293t cells were transiently transfected with hCD36 and/or hPAFR, as described in methods (A–B). Transfected cells were incubated with FITC-oxLDL (30 µg/mL) for 1 h at 37°C. Cells were washed and fixed with paraformaldehyde 4%. Fluorescence was visualized by microscopy (C). Transfected cells were stimulated with oxLDL (30 µg/mL) and the IL-8 concentration in the supernatant was measured after 24 h (D). * p<0.05 comparing with cDNA3 plasmid control transfected cells.
Figure 6 CD36 and PAFR cooperatively mediate FITC-oxLDL uptake and IL-8 production. HEK 293t cells were transiently transfected with hCD36 and/or hPAFR, as described in methods (A–B). Transfected cells were incubated with FITC-oxLDL (30 µg/mL) for 1 h at 37°C. Cells were washed and fixed with paraformaldehyde 4%. Fluorescence was visualized by microscopy (C). Transfected cells were stimulated with oxLDL (30 µg/mL) and the IL-8 concentration in the supernatant was measured after 24 h (D). * p<0.05 comparing with cDNA3 plasmid control transfected cells.CC BY · PubMed Central
Figure 7 Based on these results we conclude that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the p38 and JNK MAPK. Phosphorylation of Akt requires engagement of PAFR and a Gαi-coupled protein. This should lead to NFκB and/or AP-1 activation and gene transcription. We found increased expression of CD36, IL-8 and MCP-1 mRNA in cells stimulated with oxLDL in a PAFR-dependent manner. Moreover, we observed increased expression of CD36 protein on macrophage membrane and increased uptake of oxLDL by these cells upon exposure to oxLDL which were also dependent on PAFR. By transfecting CD36 and PAFR to HEK 293t cells, we found that whereas each receptor per se was able to induce oxLDL uptake, both receptors were required for IL-8 production. These results point to an important role for PAFR in atherosclerosis by promoting foam cells formation because of the increased uptake of oxLDL and also by contributing to macrophage infiltration due to IL-8 and MCP-1 production. Thus, antagonists of PAFR might be a promising target for atherosclerosis treatment. Dashed lines represent data obtained from the literature [21].
Figure 7 Based on these results we conclude that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the p38 and JNK MAPK. Phosphorylation of Akt requires engagement of PAFR and a Gαi-coupled protein. This should lead to NFκB and/or AP-1 activation and gene transcription. We found increased expression of CD36, IL-8 and MCP-1 mRNA in cells stimulated with oxLDL in a PAFR-dependent manner. Moreover, we observed increased expression of CD36 protein on macrophage membrane and increased uptake of oxLDL by these cells upon exposure to oxLDL which were also dependent on PAFR. By transfecting CD36 and PAFR to HEK 293t cells, we found that whereas each receptor per se was able to induce oxLDL uptake, both receptors were required for IL-8 production. These results point to an important role for PAFR in atherosclerosis by promoting foam cells formation because of the increased uptake of oxLDL and also by contributing to macrophage infiltration due to IL-8 and MCP-1 production. Thus, antagonists of PAFR might be a promising target for atherosclerosis treatment. Dashed lines represent data obtained from the literature [21].CC BY · PubMed Central

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Metadata

Authors
Rios, F. J. O., Koga, M. M., Ferracini, M., & Jancar, S.
Affiliations
Universidade de São Paulo (BR)
Corresponding author
Rios, Francisco J. O.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-05-03
Volume
7
Issue
5
Pages
e36632
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0036632
PMC
PMC3343035
OpenAlex
W2056458080
Cited by
51
References
34
Citations by year
2024: 2 · 2023: 5 · 2021: 4 · 2020: 3 · 2019: 3 · 2018: 2 · 2017: 2 · 2016: 6 · 2015: 2 · 2014: 7 · 2013: 11 · 2012: 3
FWCI
3.22
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Fundação de Amparo à Pesquisa do Estado de São Paulo (06/03982-5)
  • Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
  • Conselho Nacional de Desenvolvimento Científico e Tecnológico (2006/03982-5)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Atherosclerosis and Cardiovascular Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sphingolipid Metabolism and Signaling
Keywords
platelet-activating factor receptor, CD36, oxidized LDL, macrophage activation, PI3K/Akt signaling, MAPK signaling, interleukin-8, MCP-1, atherosclerosis, macrophage foam cell formation
MeSH terms
Chromones, Enzyme Inhibitors, Humans, Lipoproteins, LDL, Macrophage Activation, Macrophages, Models, Biological, Morpholines, Platelet Membrane Glycoproteins, Signal Transduction, Up-Regulation, Interleukin-8, Chemokine CCL2, CD36 Antigens, GTP-Binding Protein alpha Subunits, Gi-Go, Phosphatidylinositol 3-Kinases, Receptors, G-Protein-Coupled, Proto-Oncogene Proteins c-akt, HEK293 Cells

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