CD36 participates in a signaling pathway that regulates ROS formation in murine VSMCs
Wei Li, … , Masayuki Yamamoto, Roy L. Silverstein
Wei Li, … , Masayuki Yamamoto, Roy L. Silverstein
Published October 11, 2010
Citation Information: J Clin Invest. 2010;120(11):3996-4006. https://doi.org/10.1172/JCI42823.
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Research Article Cardiology

CD36 participates in a signaling pathway that regulates ROS formation in murine VSMCs

Abstract

CD36 is a membrane glycoprotein expressed on platelets, monocytes, macrophages, and several other cell types that was recently demonstrated to be involved in platelet activation in response to oxidized phospholipids, including oxidized LDL. Although the role of CD36 in other vascular cells has not been well defined, previous studies have demonstrated that cd36-knockout (cd36–/–) mice have prolonged thrombosis times after vascular injury, which can be protective in the state of hyperlipidemia. Here, we found significantly less ROS in the vessel walls of cd36–/– mice compared with WT after chemically induced arterial injury, suggesting that CD36 may contribute to ROS generation in the VSMCs themselves. Gene expression analysis revealed that the antioxidant enzymes peroxiredoxin-2 (Prdx2) and heme oxygenase-1 were upregulated in cd36–/– VSMCs. Molecular dissection of the pathway in isolated mouse VSMCs revealed CD36 ligand-dependent induction of Fyn phosphorylation, with subsequent phosphorylation and degradation of the redox-sensitive transcription factor Nrf2. Chromatin immunoprecipitation experiments further showed that Nrf2 directly occupied the Prdx2 promoter. The importance of this pathway was evidenced by increased ROS generation in prdx2–/– mice and decreased thrombosis times in both prdx2–/– and nrf2–/– mice after vascular injury. These data suggest that CD36-mediated downregulation of antioxidant systems in VSMCs may contribute to its prothrombotic, proinflammatory, and atherogenic effects.

Authors

Wei Li, Maria Febbraio, Sekhar P. Reddy, Dae-Yeul Yu, Masayuki Yamamoto, Roy L. Silverstein

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Figure 4

The transcription factor Nrf2 regulates Prdx2 expression.

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The transcription factor Nrf2 regulates Prdx2 expression. (A) ChIP assay...
(A) ChIP assays. Both cARE and eARE sites were amplified from input of WT and cd36–/– VSMCs using specific primers as indicated in Methods. An antibody to Nrf2 precipitated the cARE and eARE sites from both WT and cd36–/– VSMCs. Nonimmune IgG control antibody did not precipitate these sites. (B) Schema of the Prdx2 promoter regions examined by luciferase expression assay. An 869-bp region from the Prdx2 promoter that includes the cARE, eARE, and eARE-like sites (P6) as well as truncated constructs containing only the cARE (P1) or eARE (P3) sites, the cARE or eARE with the eARE-like sites (P4 and P5), or the eARE-like sites without cARE or eARE sites (P2) were cloned into a luciferase reporter vector, pGL2-Basic. 3 additional constructs (M1–M3) were generated in which sets of 3 adjacent nucleotide triplets within the eARE site were mutated to TTT. (C) Luciferase reporter activity of Prdx2 promoter sequence. Plasmid vectors containing the Prdx2 promoter sequences were transfected into the WT VSMCs and luciferase activity examined 48 hours later. The luciferase signals were adjusted by protein concentration and expressed as fold change compared with basal levels. *P < 0.0001 vs. B, P1, P2, P4, and M1–M3; #P < 0.001 P6 vs. P3 and P5. (D) Nrf2 cDNA transfection induces Prdx2 expression in VSMCs. Plasmid vector pcDNA3 encoding mouse Nrf2 cDNA (pcDNA3-mNrf2) or empty vector was transfected into WT VSMCs, and expression of Nrf2 and Prdx2 were quantified by Western blot. One-way ANOVA (Bonferroni/Dunn) was used to determine the differences among groups. Data are represented as mean ± SEM.