BRG1 Activates PR65A Transcription to Regulate NO Bioavailability in Vascular Endothelial Cells

Abstract

Vascular endothelial cells contribute to the pathogenesis of cardiovascular diseases by producing and disseminating angiocrine factors. Nitric oxide (NO), catalyzed by endothelial NO synthase (eNOS), is one of the prototypical angiocrine factors. eNOS activity is modulated by site-specific phosphorylation. We have previously shown that endothelial-specific knockdown of BRG1 in Apoe ^-/- mice attenuates the development of atherosclerosis, in which eNOS-dependent NO catalysis plays an antagonizing role. Here we report that attenuation of atherogenesis in mice by BRG1 knockdown was accompanied by partial restoration of NO biosynthesis by 44% in the arteries and a simultaneous up-regulation of eNOS serine 1177 phosphorylation by 59%. Indeed, BRG1 depletion or inhibition ameliorated oxLDL-induced loss of NO bioavailability and eNOS phosphorylation in cultured endothelial cells. Further analysis revealed that BRG1 regulated eNOS phosphorylation and NO synthesis by activating the transcription of protein phosphatase 2A (PP2A) structural subunit a (encoded by PR65A). BRG1 interacted with ETS1, was recruited by ETS1 to the PR65A promoter, and cooperated with ETS1 to activate PR65A transcription. Finally, depletion of ETS1, similar to BRG1, repressed PR65A induction, normalized eNOS phosphorylation, and rescued NO biosynthesis in endothelial cells treated with oxLDL. In conclusion, our data characterize a novel transcriptional cascade that regulates NO bioavailability in vascular endothelial cells.

Keywords: BRG1; PP2A; eNOS; nitric oxide; phosphatase; phosphorylation; transcriptional regulation; vascular endothelial cells.

FIGURE 1

BRG1 regulates NO bioavailability by modulating eNOS phosphorylation in mice. 8-week male Apoe^–/– mice were injected with lentivirus carrying endothelial-specific BRG1 shRNA (Endo-shBRG1) or the control virus (shC) and fed a Western diet or a control diet for 8 weeks as described in “Materials and Methods.” (A) Aortic arteries were homogenized and NO content was examined using the Griess assay. (B,C) Gene expression levels in the aortic arteries were examined by qPCR and Western. N = 5 mice for the control diet groups and N = 7 mice for the Western diet groups. *p < 0.05.

FIGURE 2

BRG1 regulates NO bioavailability by modulating eNOS phosphorylation in cultured endothelial cells. (A–C) EAhy926 cells and HAECs were transfected with siRNA targeting BRG1 or scrambled siRNA (SCR) followed by treatment with oxLDL (10 μg/ml) for 24 h. NO content in the culture media was examined by the Griess assay. Gene expression levels were examined by qPCR and Western. (D–F) EAhy926 cells and HAECs were treated with oxLDL (10 μg/ml) in the presence or absence of PFI-3 (5 μM) for 24 h. NO content in the culture media was examined by the Griess assay. Gene expression levels were examined by qPCR and Western. *p < 0.05.

FIGURE 3

BRG1 mediates PP2A transcription. (A,B) 8-week male Apoe^–/– mice were injected with lentivirus carrying endothelial-specific BRG1 shRNA (Endo-shBRG1) or the control virus (shC) and fed a Western diet or a control diet for 8 weeks as described in “Materials and Methods”. Expression levels of specific genes in the aortic arteries were examined by qPCR and Western. N = 5 mice for the control diet groups and N = 7 mice for the Western diet groups. (C,D) EAhy926 cells and HAECs were transfected with siRNA targeting BRG1 or scrambled siRNA (SCR) followed by treatment with oxLDL (10 μg/ml) for 24 h. Gene expression levels were examined by qPCR and Western. (E,F) EAhy926 cells and HAECs were treated with oxLDL (10 μg/ml) in the presence or absence of PFI-3 (5 μM) for 24 h. Gene expression levels were examined by qPCR and Western. *p < 0.05.

FIGURE 4

PP2A over-expression circumvents BRG1 deficiency to suppress NO production. (A,B) EAhy296 cells were transfected with siRNA targeting BRG1 in the presence or absence of exogenous HA-tagged PR65α followed by treatment with oxLDL (10 μg/ml) for 24 h. NO content in the culture media was examined by the Griess assay. eNOS phosphorylation was examined by Western. (C,D) EAhy296 cells were transfected with exogenous HA-tagged PR65α followed by treatment with oxLDL (10 μg/ml) and PFI-3 (5 μM) for 24 h. NO content in the culture media was examined by the Griess assay. eNOS phosphorylation was examined by Western. *p < 0.05.

FIGURE 5

BRG1 interacts with ETS1 to regulate PR65A transcription. (A) PR65A promoter-luciferase constructs were transfected into EAhy926 cells with or without BRG1 followed by treatment with oxLDL (10 μg/ml). Luciferase activities were normalized by protein concentration and GFP fluorescence. (B) EAhy926 cells and HAECs were treated with oxLDL (10 μg/ml) and harvested at indicated time points. ChIP assays were performed with anti-BRG1 or IgG. (C) Wild type and mutant PR65A promoter-luciferase constructs were transfected into EAhy926 cells with or without BRG1 followed by treatment with oxLDL (10 μg/ml). Luciferase activities were normalized by protein concentration and GFP fluorescence. (D) FLAG-tagged BRG1 and V5-tagged EST1 were transfected into HEK293 cells. Immunoprecipitation was performed with anti-FLAG. (E) Nuclear lysates extracted from EAhy926 cells were immunoprecipitated with anti-BRG1. (F) EAhy926 cells and HAECs were with oxLDL (10 μg/ml) for 24 h. Re-ChIP assays were performed with indicated antibodies. *p < 0.05.

FIGURE 6

ETS1 regulates NO bioavailability in endothelial cells. (A–C) EAhy296 cells and HAECs were transfected with siRNA targeting ETS1 followed by treatment with oxLDL (10 μg/ml) for 24 h. NO content in the culture media was examined by the Griess assay. Gene expression levels were examined by qPCR and Western. (D) A schematic model. *p < 0.05.