Overexpression of MECP2 attenuates cigarette smoke extracts induced lung epithelial cell injury by promoting CYP1B1 methylation

Abstract

MECP2 (Methyl-CpG-binding protein 2) has been shown to have a critical role in regulating DNA methylation against smoke exposed lung injury. However, the biological function of MECP2 and the underlying molecular mechanism remains elusive. Human bronchial epithelial (16HBE) and alveolar type II epithelial cells (AECII) were exposed to increasing concentrations of cigarette smoke extracts (CSE) solution to establish CSE-induced lung epithelial cell injury models. Our findings revealed that MECP2 was down-regulated, while CYP1B1 was up-regulated in CSE-induced lung epithelial cell injury models by quantitative real time PCR, western blotting and immunofluorescence staining. Down-regulated CYP1B1 was ascribed to the demethylation of its promoter by methylation-specific PCR (MSP). The in vitro experiments further showed that MECP2 overexpression significantly attenuated CSE-triggered cell growth attenuation, cell cycle arrest, apoptosis and ROS generation in lung epithelial cells by CCK-8 and flow cytometry assays. In molecular level, we further demonstrated that MECP2 overexpression obviously suppressed the expression of CYP1B1 through enhancing DNA methylation. Therefore, our data suggest that MECP2 protects against CSE-induced lung epithelial cell injury possibly through down-regulating CYP1B1 expression via elevating its methylation status.

Cellular and Molecular Signatures of Oxidative Stress in Bronchial Epithelial Cell Models Injured by Cigarette Smoke Extract

“…More specifically, in 16HBE, exposure to CSE downregulates MECP2 (Methyl-CpG-binding protein 2) and upregulates cytochrome family 1 subfamily B polypeptide 1 (CYP1B1), a member of the CYP1 family gene whose transcription is strongly modulated by DNA methylation of the promoter site. Increased methylation of CYP1B1 promoter has been observed in CSE-exposed 16HBE [72]. Of note, CSE-induced cell injury and increased methylation of CYP1B1 promoter were reversed by overexpression of MECP2 [72].…”

“…In this case, the dyes are non-fluorescent when reduced, but become highly fluorescent upon oxidation. The most widely used dye is the 2 ,7 -dichlorofluorescin diacetate (DCFDA) that become oxidized into 2 ,7 -dichlorofluorescein (DCF) by intracellular ROS [11,14,22,26,28,29,32,33,35,42,45,50,68,72,[83][84][85][87][88][89]104]. Other commercially available fluorogenic probes are also available [13,24,69,80].…”

“…Increased methylation of CYP1B1 promoter has been observed in CSE-exposed 16HBE [72]. Of note, CSE-induced cell injury and increased methylation of CYP1B1 promoter were reversed by overexpression of MECP2 [72]. Concerning histone modification, exposure of 16HBE cells to CSE decreased HDAC3 activity/expression and HDAC2 activity, contributing to steroid resistance [78,79,89].…”

“…Epigenetic alterations include chromatin remodeling, DNA methylation and PTMs of histone proteins. Several reports have shown that exposure of bronchial epithelial cells to CSE causes a number of epigenetic modifications [72,78,79,89,114]. More specifically, in 16HBE, exposure to CSE downregulates MECP2 (Methyl-CpG-binding protein 2) and upregulates cytochrome family 1 subfamily B polypeptide 1 (CYP1B1), a member of the CYP1 family gene whose transcription is strongly modulated by DNA methylation of the promoter site.…”

“…Besides affecting apoptosis and promoting cell senescence, it has been reported that CSE promotes other inflammatory forms of cell death, such as the pyroptosis or other forms of caspase-dependent cell deaths [12,14,15,27,28,[30][31][32][33][34]68,69,72,73,105]. Pyroptosis is a type of pro-inflammatory cell death promoted by caspase-1 activation and characterized by cell lysis and interleukin IL-1β release.…”