Effect Of Transcription Factor Sp1 On The Regulation Of Key Genes In Cholesterol Reversal

SR-BI is one of the key proteins in the process of reverse cholesterol transport, and its major function is to selectively uptake HDL cholesterol from plasma into liver cells and then to regulate HDL-C level in the plasma. Therefore, the regulation of SR-BI expression is important for controlling blood lipid levels and reducing the risks of cardiovascular diseases.In the first part of this study, we found that SR-BI expression was significantly increased in the liver of ApoE-/- mice on high fat diet and in hepatic cells HepG2 on low density lipoprotein (LDL) treatment. In order to study the mechanism of SR-BI activation, we found that the transcription factor Sp 1 plays a key role in this process by using SR-BI gene promoter luciferase reporters. When Sp1 was knocked down by Spl-siRNA, LDL would not activate SR-BI expression, whereas overexpression of Sp1 gene had no obvious effect on the activation in HepG2 cells. A series of co-immunoprecipitation experiment results showed that the activation of SR-BI was associated with the change of Sp1 protein complexes formed in the promoter region of SR-BI, where histone acetyltransferase p300 was recruited and histone deacetylase HDAC1 was separated. As a result, histone acetylation increased, leading to activation of gene transcription. After further investigation, we found that in the presence of LDL, the Sp1 protein post-translational modification changed, i.e., Sp1 phosphorylation increased. Through the analysis of the HepG2 cell phosphorylation pathway in the presence of LDL, we found that ERK1/2 kinase phosphorylation level increased. LDL could not cause phosphorylation of Sp1 and activation of SR-BI when cells were treated with ERK signaling inhibitor U1026. The results together showed that Sp1 was phosphorylated through ERK 1/2 pathway and then activated SR-BI when cells were treated with LDL. In order to study the relationship between Spl phosphorylation and Sp1 protein complex change, we constructed the eukaryotic expression plasmid pCMV-Flag-Spl, and analyzed the modification sites of Sp1 by biological mass spectrometry. The results showed that a number of phosphorylation sites were examined, including a new Sp1 phosphorylation site Ser702. According to the relevant literatures, this phosphorylation site of Sp1 might influence its binding ability to HDAC1. After construction of 702 serine mutant plasmid pCMV-Flag-Spl, we observed that the recruitment of HDAC1 by mutated Sp1 would not be affected by LDL. These results demonstrated that Sp1 protein serine 702 phosphorylation was involved in the activation process in SR-BI by LDL.In the second part of this study, liver proteomics of ApoE-/- mice on high fat diet were analyzed using a label-free quantitative proteomic method based on gel-LC-MS/MS. The results showed that the levels of total cholesterol and LDL-C in ApoE-/- mice on high fat diet increased significantly. Through mass spectrometry data analysis, we identified 6677 proteins and 571 differences, and these proteins were mainly involved in metabolism, transport and other biological processes in GO classification results. Expression of proteins in catabolic process increased and expressions of protein in biosynthetic process decreased. KEGG analysis showed that, expression of PI3K-Akt pathway related protein increased in mouse with high fat diet.