Class II transactivator (CIITA) mediates IFN-γ induced eNOS repression by enlisting SUV39H1

https://doi.org/10.1016/j.bbagrm.2019.01.005Get rights and content

Highlights

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    IFN-γ induced CIITA up-regulation and eNOS down-regulation in endothelial cells.

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    CIITA mediates IFN-γ induced eNOS trans-repression.

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    CIITA interacts with and recruits SUV39H1 to the eNOS promoter.

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    SUV39H1 is essential for IFN-γ induced eNOS trans-repression.

Abstract

Endothelial nitric oxide synthase (eNOS), selectively expressed in vascular endothelial cells, plays important roles in a range of biological and pathological processes. eNOS levels can be altered by extrinsic and intrinsic cues at the transcriptional level. Here we examined the epigenetic mechanism whereby the pro-inflammatory cytokine interferon gamma (IFN-γ) represses eNOS transcription. In response to IFN-γ treatment, there was a simultaneous down-regulation of eNOS expression and up-regulation of class II trans-activator (CIITA). Over-expression of CIITA directly repressed eNOS promoter while CIITA knockdown attenuated IFN-γ induced eNOS repression. Chromatin immunoprecipitation (ChIP) assay revealed that IFN-γ stimulation promoted CIITA occupancy on the proximal eNOS (−430/−168). Coincidently, CIITA recruitment to the eNOS promoter was paralleled by the disappearance of trimethylated histone H3K4 (H3K4Me3) and the enrichment of trimethylated H3K9 (H3K9Me3) with no significant changes in the levels of trimethylated H3K27 (H3K27Me3) or trimethylated H4K20 (H4K20Me3). In accordance, CIITA depletion was associated with the normalization of H3K4Me3 and H3K9Me3 on the eNOS promoter. Mechanistically, CIITA interacted with and enlisted the histone H3K9 trimethyltransferase SUV39H1 to the eNOS promoter to repress transcription. IFN-γ treatment augmented SUV39H1 expression and promoted SUV39H1 recruitment to the eNOS promoter in endothelial cells. Silencing of SUV39H1 abrogated eNOS repression by IFN-γ by erasing H3K9Me3 from the eNOS promoter. In conclusion, our data reveal a novel role for CIITA in endothelial cells and present SUV39H1 as a druggable target in the intervention of endothelial dysfunction.

Introduction

Endothelial cells constitute a unique source of humoral factors that may regulate the functions of other cell types via paracrine or endocrine pathways [1]. Among the many molecules originated from the endothelium, nitric oxide (NO) plays versatile roles in maintaining the internal homeostasis. NO promotes relaxation of smooth muscle cells [2], suppresses inflammatory reaction in macrophages and adipose tissue [3], and sensitizes skeletal muscle cells to insulin [4]. In accordance, NO deficiency is associated with an array of human diseases including hypertension, type 2 diabetes, and atherosclerosis [5].

NO is synthesized in the endothelium by endothelial NO synthase (eNOS). Therefore, fluctuations of NO levels under various physiological and pathophysiological conditions reflect the changes in eNOS expression or activity. For instance, eNOS expression is markedly up-regulated in pulmonary vessels right after birth allowing sufficient NO discharge and pulmonary vasodilation [6]. On the other hand, turbulent shear stress, which occurs more frequently at the aortic arch, serves to down-regulate eNOS transcription and promote atherogenesis [7]. Similarly, aging contributes to cardiovascular diseases in part by repressing eNOS transcription [8]. Consistent with this notion, transcription modulators, including KLF2, AP-1, Sp1, and Ets-1, participate in the pathogenesis of human diseases by regulating eNOS expression [9]. We have previously shown that myocardin-related transcription factor A, or MRTF-A, mediates transcriptional repression of eNOS in vascular endothelial cells exposed to oxidized low-density lipoprotein (oxLDL), a known risk factor for atherosclerosis [10]. Systemic MRTF-A-null mice are protected from atherosclerosis although it is unknown whether this phenotype is underscored by eNOS normalization in endothelial cells [11].

Interferon gamma (IFN-γ) is a prototypical pro-inflammatory cytokine primarily released by CD4+ Th1 lymphocytes [12]. Usually functioning within the adaptive immune system to safeguard the organism from invading pathogens, excessive IFN-γ production is thought to be associated with cardiovascular diseases [13], metabolic disorders [14], and neurodegenerative diseases [15]. Recently it has been demonstrated that angiotensin II induced hypertension was attenuated in mice with a deficiency in interferon gamma (IFN-γ) [16]. We therefore hypothesized that IFN-γ may contribute to hypertension by influencing eNOS levels in vascular endothelial cells. Our data as summarized here suggest that IFN-γ directly represses eNOS transcription. Class II trans-activator (CIITA) mediates IFN-γ induced eNOS repression by enlisting the histone H3K9 trimethyltransferase SUV39H1. Therefore, targeting the CIITA-SUV39H1 axis may yield novel interventional strategies for vascular endothelial disorders.

Section snippets

Cell culture and treatment

Human primary aortic endothelial cell (HAEC) was purchased from Lonza (Heidelberg, Germany) and maintained in were maintained in EGM-2 media with supplements supplied by the vendor as previously described [17,18]; experiments were performed in HAECs between 3rd and 6th passages. Immortalized human umbilical vein endothelial cell (HUVEC/EAhy926) and human embryonic kidney cell (HEK293) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine

IFN-γ represses eNOS transcription in vascular endothelial cells

We started by examining the effect of IFN-γ treatment on eNOS transcription in vascular endothelial cells. Exposure of immortalized endothelial cells (EAhy926) to IFN-γ led to a reduction in eNOS mRNA (Fig. 1A) and protein (Fig. 1B) levels starting at 12 h post-treatment. The down-regulation of eNOS levels continued with time when eventually they were repressed by ~50% at 48 h. We also performed similar experiments with human primary aortic endothelial cells (HAECs). Quantitative PCR (Fig. 1C)

Discussion

Alteration of eNOS expression serves as both a biomarker and a potential mechanism underlying a host of cardiovascular diseases [36]. Here we detail a novel epigenetic mechanism underlying the transcriptional regulation of the eNOS gene by the pro-inflammatory cytokine IFN-γ in endothelial cells. Our data demonstrate that class II trans-activator interacts with the histone methyltransferase SUV39H1 to mediate IFN-γ induced eNOS repression (Fig. 6G).

CIITA is mostly known as a transcriptional

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Acknowledgements

This work was supported, in part, by grants from the National Natural Science Foundation of China (81670223, 81870302, 81570420).

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