Oxysterol Regulates Oxysterol Binding Protein Like 2(OSBPL2)via The P53/SREBF2/NFYA Signaling Pathway

Background:Oxysterol binding protein(OSBP),a kind of receptor protein and has high affinity with oxysterols.In addition,proteins with high homology to the OSBP are called OSBP-like proteins(OSBPLs)or OSBP-related proteins(ORPs),which can also bind oxysterols.The structures and functions of OSBPLs are diverse,and their main functions are similar to that of OSBP and can be used as transporters for transporting oxysterol on the cytoplasm,vesicles,and Golgi membranes.The human OSBPLs family include 12 members and there are extensive splicing variations.One member of this conserved family is called Oxysterol Binding Protein Like 2(OSBPL2),and the biological significance of OSBPL2 is currently focused on sterol transport and metabolic regulation.Previous studies have shown that silencing of OSBPL2 leads to a 1.9-fold increase in cellular 25-hydroxycholesterol(25-OHC),which regulates the expression of lipid metabolism related genes.Our study found that exogenous 25-OHC in the cells resulted in lower expression of OSBPL2.Therefore,we hypothesize that there are certain negative feedback loop regulation,and the purpose of this study is to investigate the relationship between 25-OHC and OSBPL2.Methods:(1)HeLa cells were treated with different concentrations of 25-OHC and the expression of OSBPL2 was detected;(2)Determining the promoter region of OSBPL2 via bioinformatics and others' methods;(3)Cloning and initial identification of the promoter region of OSBPL2;(4)Constructing serials of truncated vectors to determine the key region of OSBPL2 promoter;(5)Chromatin immunoprecipitation assay(ChIP)was used to identify transcriptional factor;(6)Constructing the overexpression and deletion mutations,RNA interference were used to determine the core transcription factor binding region;(7)Transcriptome sequencing analysis was carried out to find target genes and related signaling pathways.Results:(1)25-OHC down-regulated the expression of the OSBPL2 gene;(2)Dual-Luciferase Reporter Assay showed that a core functional promoter was present in the-109 to+110bp region.As predicted by bioinformatics,the core functional promoter included several transcription factor-binding sites,such as PLAG1 zinc finger(PLAG1),transcription factor AP-2 alpha(TFAP2A),nuclear transcription factor Y subunit alpha(NFYA),nuclear respiratory factor 1(NRF1)and ETS transcription factor(ELK4);(3)Deletion and overexpression of transcription factors suggested NFYA and PLAG1 were essential for basal transcriptional regulation of OSBPL2,however,when 25-OHC was added,the promoter activity of the NFYA-knockdown group was significantly lower than the PLAG1-knockdown group.And ChIP assay indicated that NFYA binds to CCAAT in the OSBPL2 promoter in vivo.(4)Next generation RNA sequencing showed that the most up-regulated mRNAs included tp53 whereas one of the most down-regulated individual mRNAs was SREBF2.Then we verified that those targets actually participates in transcriptional regulation of OSBPL2 by 25-OHC.So we believe that the p53/SREBF2 signaling pathway was involved in regulation of 25-OHC-induced OSBPL2 expression.Conclusions:We first identified that NFYA and PLAG1 were important for the basal transcription of OSBPL2.We also show that 25-OHC inhibited·NFYA-dependent transcription of OSBPL2 via the p53/SREBF2 pathway.The present study provides a new insight into the relationship between 25-OHC and OSBPL2.