Acetylation Of NF-?B Mediates The Regulation Of DDAH1 Gene Expression Under Hypoxic Conditions

Introduction:Cardio-cerebrovascular disease?CCVD?is a kind of multifactorial disease caused by genetic and environmental factors,and it has become a common cause of death in the word.The pathological changes of blood vessels such as the lipid deposition of vascular wall,chronic inflammatory response and vascular smooth muscle hyperplasia can lead to local tissue ischemia and hypoxia.Studies have found that hypoxia can lead to an increase in the production of NO which plays an important role in vasodilation.NO is a major regulator of vasomotor function and plays a key role in intravascular environmental stability.NO is produced by the nitric oxide synthase?NOS?enzyme.Asymmetric dimethylarginine?ADMA?is an endogenous NOS inhibitor that is a risk factor for cardiovascular mortality and progression of chronic kidney disease.ADMA in the circulation is mainly degraded by dimethylarginine dimethylaminohydrolase?DDAH?.There are two subtypes of DDAH,including DDAH1 and DDAH2.Studies have shown that DDAH1 is the key enzyme that degrades ADMA,and there are several sites of lipid metabolism and inflammation-related transcription factors in the DDAH1promoter region.NF-?B is a nuclear transcription factor involved in mediating the inflammatory response of cells.Hypoxia causes inflammatory reaction of tissues in varying degrees.At the same time,NF-?B binds to target DNA and triggers a series of pro-inflammatory reactions under hypoxic conditions.The acetylation of NF-?B enhances its binding to DNA and promoted gene transcription.Our group has previously confirmed that NF-?B participated in the regulation of the expression of nNOS and DDAH2 genes through acetylation.In this study,the binding sites of NF-?B in the promoter region of DDAH1 was predicted by bioinformatics software JASPAR and Alibaba2,and CoCl₂ was used to mimic hypoxic conditions.The expression level of DDAH1 and the binding of transcription factors in cells were determined by relevant molecular biology techniques.At the same time,TSA was used to stimulate SH-SY-5Y and HEK293 cells to promote the acetylation of related proteins,and identified the regulation mechanism of NF-?B of DDAH1 under hypoxia.Our studies may elucidate a possible molecular mechanism of cardio-cerebrovascular disease,and provide a new strategy for the treatment of cardio-cerebrovascular disease.Materials and methods:Materials1.Human embryonic kidney cell line HEK2932.Human neuroblastoma cell line SH-SY-5Y3.Real-time PCR and Western Blot related reagents4.Immunoprecipitation related reagents5.Luciferase vector construction and testing related reagents6.ChIP and DNA binding assays related reagents Materials1.HEK293 and SH-SY-5Y cell lines were treated with CoCl₂?mimic hypoxia?,TSA?inhibition of deacetylase?,and both respectively,and the expression levels of DDAH1 at different times were detected by Real-time PCR and Western blot.2.Luciferase reporter vectors were constructed and the promoter activity was assessed by luciferase assay under normoxia,hypoxia,acetylation,or hypoxia with acetylation.3.The binding of NF-?B and DDAH1 promoter region in vivo and in vitro was verified by ChIP and DNA binding assays,respectively.4.IP and Western Blot assays were used to detect the acetylation level of NF-?B in SH-SY-5Y cell line under different conditions.Results:1.HEK293 and SH-SY-5Y cells,were treated with CoCl₂?100?M?to mimic hypoxia.Real-time PCR and Western blot assays were performed to detect the expression level of DDAH1 in 0h,and 6h,12h,24h after CoCl₂ treatment.The results showed that hypoxia can upregulate the mRNA and protein expression levels of DDAH1..2.JASPAR and ALIBABA2 analysis showed a potential NF-?B binding site in the promoter region of DDAH1,which was located at-1273 to-1282 of the upstream of the translation initiation site.3.Dual luciferase reporter assay results showed that the luciferase activity in the pGL-1291 group was significantly higher than that in the pGL-3 and pGL-1194 groups?P<0.05?;After using hypoxia,acetylation,and their combined treated cells,the luciferase activity of pGL-1291 increased significantly?P<0.05?.4.In ChIP assays,chromatin immunoprecipitation was performed with NF-?B antibody,and DNA precipitates were amplified by PCR.The results of agarose gel electrophoresis showed DNA bands of 138 bp,demonstrating that NF-?B bound to the DDAH1 promoter in vivo.5.In the DNA binding assays,the non-labeled DNA probe set didn't show any bands.While the biotin-labeled DNA probe set formed the same band as the input,proving that NF-?B bound to the NF-?B response element in DDAH1 promoter in vitro.6.The acetylation level of NF-?B with the treatment of Co Cl₂,TSA or both was detected by IP and Western Blot.The results showed that the acetylation of NF-?B increased under all the above three conditions.?P<0.01?7.HEK293 and SH-SY-5Y cells were stimulated with TSA?250ng/mL?.Real-time PCR and Western Blot assays were used to detect the expression of DDAH1 under 0h,6h,12h,24h treatment.The results showed that acetylation can upregulate the mRNA and protein expression levels of DDAH1.8.HEK293 and SH-SY-5Y cells were co-treated with CoCl₂?100?M?and TSA?250ng/mL?.Real-time PCR and Western Blot results showed that hypoxia combined with acetylation can upregulate the mRNA and protein expression levels of DDAH1.Conclusions:1.DDAH1 is one of the target genes of NF-?B.2.NF-?B participates in the regulation of DDAH1 gene expression under hypoxic conditions through acetylation.