Construction Of Pcdna-sm22-SCAP(D443N) Eukaryotic Expression Vector And Its Investigation Of Function

Cholesterol is derived from two pathways, one is the uptake of plasma via LDL receptor (LDLr)-mediated endocytosis, and the other is from cellular synthesis. In the pathway of cholesterol synthesis, HMGCoA reductase is the important rate-limiting enzyme. Either HMGCoA reductase or LDLr activity is under control via a feedback system that is dependent on intracellular cholesterol concentration. SREBP2 is a three-domain protein that is bound to membranes of the endoplasmic reticulum and the nuclear envelope. The NH2-terminal domain of SREBP2 is a transcription factor that binds to sterol-regulatory element found in promoters of multiple genes encoding enzymes crucial in sterol biosynthesis and sterol uptake. Proteolytic release of the NH2-terminal domain of the SREBPs is processed by sequential cleavages. The proteases cleaving the SREBPs are activated by SREBP-cleacage-activating protein (SCAP). SCAP-SREBP complexes are generated when intracelluar sterol levels are decreased. SCAP harbours a sterol-sensing domain that, upon increased levels of cellular cholesterol, inhibits complex formation, thus creating a negative feedback loop.Objective: The experiment was designed to investigate the function of SREBP cleavage-activating protein (SCAP) mutant (D443N) by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 (pGL3-SM22-SCAP(D443N)). The effect of SCAP gene overexpression on lipid metabolism were investigated in CHO,HMCL and VSMCs.Methods: 1.SM22 promoter (pSM22) amplified from genome DNA of mice by nested PCR, and SCAP(D443) mutant amplified from plasmid pTK-HSV-SCAP(D443N) were cloned into pcDNA3.1 to construct pcDNA-SM22-SCAP(D443N).2.Primary culture VSMCs was producted by two weeks mice.3.We detected the expression and function of plasmid by transfected it into CHO,HMCL and VSMCs. Intracellular cholesterol content, mRNA and protein expression of SCAP, SREBP2, LDLr and HMGCoA reductase in the treated HMCs were assessed by Oil Red O staining, cholesterol enzymatic assay, real-time quantitative polymerase chain reaction, Western bloting analysis, immunohistochemical test.Results:1.Recombinant pcDNA-SM22-SCAP(D443N) was successfully constructed.2.Primary culture of VSMCs was completed.3.We demonstrated that SCAP overexpression enhanced transformation of HMCs into foam cells by increasing uptake of unmodified LDL via LDLr as evidenced by Oil Red O staining and direct assay of intracellular cholesterol. LDL decreased LDLr, SREBP2, HMGCoA mRNA and protein expression at physiological condition. However, SCAP overexpression enhanced LDLr expression, overriding the suppression of LDLr induced by LDL and inappropriately increasing LDL uptake. SCAP over-expression of mRNA and protein of HMGCoA and SREBP2. These observations indicate that SCAP overexpression disrupts cholesterol-mediated LDLr feedback regulation, permitting intracellular accumulation of unmodified LDL and causing foam cell formation.Conclusion: The pGL3-SM22-SCAP(D443N) eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.The implication of the plasmid function findings is that lipid accumulation in cells may be caused by SCAP overexpression.