Alkaline Ceramidase 2 Is A Novel Direct Target Of P53 And Induces Autophagy And Apoptosis Through Enhancing ROS Generation

ACER2 is a Golgi membrane protein with several putative transmembrane domains(TMDs)and is widely expressed in a variety of normal tissues except the placenta.In response to a variety of cellular stimuli such as DNA damage,serum starvation will be significantly up-regulated.ACER2 involved in sphingomyelin metabolism,can catalyze the hydrolysis of ceramide to sphingosine,sphingosine further phosphorylation to produce 1 sphingosine phosphate.ACER2 can control the fate of cells by controlling the relative levels of ceramide,sphingosine and sphingosine 1-phosphate,and then in the process of cell proliferation,senescence and apoptosis.The ACER2 transcription initiation site has been identified and the ACER2 gene promoter has been identified to be located 670 bp upstream of the transcription initiation site.The results of transcription factor binding site analysis showed that the ACER2 gene promoter contained potential transcription factor binding sites such as Sp1,GATA-1 and AP-1.In this study,we will further analyze the important cis-acting elements in the ACER2 promoter region,and further study the function and mechanism of ACER2 in the process of cellular life.(1)Further analysis of important transcription factor binding sites in the ACER2 promoter regionThe genomic structure and gene composition of ACER2 were analyzed by UCSC genome browser.The ACER2 gene was found to be transcribed according to ENCODE chromosome modification data and ChromHMM chromosome state segmentation data.The promoter region of ACER2 was subjected to transcription factor binding site analysis by using the MatInspector Professional Edition and TFSEARCH software and found have two potential p53 binding sites.These two potential p53 binding site sequences are conserved in human,rat and mice,suggesting that they play an important role in the transcriptional regulation of the ACER2 promoter.(2)p53 directly regulates the transcription of the ACER2 promoterIn the first place,a series of the ACER2 promoter constructs and three mutants were constructed which have mutated binding sites for p53 and the p21 and Bax reporter genes were transfected separately or co-transfected with p53 plasmid into H1299 cells.The results showed that p53 overexpression could significantly increase the activity of reporter gene of p21 and Bax and the activity of ACER2-P397,ACER2-P676,ACER2-P800 and ACER2-P1285,but the up-regulation of the promoter activity of the first p53-binding site mutation was significantly attenuated or even lost with p53 overexpression.These suggest that p53 plays a direct role in ACER2 direct activation through the p53 binding site in the core region of the ACER2 promoter.At the same time,the p53 overexpression plasmid was transfected into H1299 cells.The results of quantitative QRT-PCR showed that overexpression of p53 could lead to the increase of endogenous ACER2 mRNA level.The results of quantitative QRT-PCR showed that DNA damage could increase the mRNA level of endogenous ACER2 in p53 wild type A549 cells.Then siRNA transfected into A549 cells and then treated with ADR,quantitative QRT-PCR results show that siRNA-mediated inhibition of p53 expression can lead to cell endogenous ACER2 mRNA levels decreased.In addition,the chromatin immunoprecipitation assay(ChIP)test results also confirmed that p53 can bind the first potential p53 binding site in the core region of the ACER2 promoter.These results suggest that p53 can directly regulate the transcription of the ACER2 promoter and ACER2 is a novel p53 target gene.(3)ACER2 can induce autophagy and apoptosisFirst,the ACER2 eukaryotic expression plasmid and the p53 expression plasmid were transfected into H1299 cells alone or in combination.The results of wetern blot analysis showed that ACER2 overexpression could up-regulate the expression of LC3-II and can induce stronger LC3-II up-regulation co-transformation with p53.The results of transmission electron microscopy(TEM)showed that ACER2 overexpression could induce the increase of autophagosomes and induce more autophagosomes in combination with p53.Immunofluorescence and transfection of GFP-LC3 analysis showed that ACER2 overexpression could induce the increase of LC3-II spots,and the co-transformation with p53 could induce more LC3-II spots.The results of flow cytometry analysis showed that ACER2 overexpression in H1299 cells increased the cell apoptosis,and combined with p53 will help p53 induce more apoptosis.The ACER2 expression plasmid was transfected into A549 cells and stimulated with ADR.The results of western blot and flow cytometry analysis showed that ACER2 was helpful for autophagy and apoptosis induced by DNA damage.Then we transfected ACER2 siRNA and autophagy key gene ATG5 siRNA into A549 cells,combined with ADR treatment,western blot and flow cytometry analysis results showed that ACER2 is essential for DNA damage induced autophagy and apoptosis.Then we removed the deletion of 13 and 36 amino acid residues at the N-terminus of ACER2 protein by site-directed mutagenesis PCR and the two deletions were transfected into H1299 cells.The results of western blot analysis showed that deletion of N-terminal 13,36 amino acid residues are also able to induce autophagy.These results suggest that ACER2 can induce autophagy and apoptosis,and contribute to p53 and DNA damage induced autophagy and apoptosis.(4)ACER2 induces autophagy and apoptosis through ROSWe used NAC to treat the cells and then the ACER2 expression plasmid was transfected into H1299 cells.The results of flow cytometry analysis showed that NAC could clear the ROS produced by ACER2 overexpression.Then we examined the autophagy and apoptosis by Western blot and flow cytometry,and found that ACER2-induced autophagy and apoptosis were significantly attenuated after treatment with NAC.We then transfected ACER2 expression plasmid into H1299 and then treated with H2O2.Western blot and flow cytometry analysis showed that ACER2 was helpful for H2O2-induced autophagy and apoptosis.These results suggest that ACER2 induces apoptosis and autophagy by inducing ROS generation.In summary,our study not only found that the ACER2 promoter region contains important transcription factor binding sites such as "star gene" p53,and further clarifies that p53 can directly activate the transcription of the ACER2 promoter and can regulate endogenous ACER2 expression.Transcriptional activation of ACER2 can induce autophagy and apoptosis,and contribute to p53 and DNA damage induced autophagy and apoptosis.DNA damage-induced autophagy and apoptosis require ACER2 gene involvement.It is clear that ACER2 can produce more ROS by the accumulation of sphingosine to autophagy and apoptosis.In this study,we further study the regulation of ACER2 promoter transcriptional regulation in the process of cell autophagy and apoptosis and the molecular mechanism of tumor development.Our study also has important scientific significance in the regulation of p53 on the sphingomyelin metabolism as well as molecular oncology theory.