Effect Of Cobaltous Chloride On The Transcription Activity Of Modified Human ENOS Promoter

AIM:To construct modified promoter of human endothelial nitric-oxide synthase(eNOS) and investigate transcriptional activity of the eNOS promoter via a hypoxia model of human umbilical vein endothelial cells-12(HUVEC-12) stimulated by Cobaltous chloride in vitro. METHODS:The eucaryotic expression vector of pGL2-eNOS-repSP3 with promoter of eNOS gene was constructed by DNA site-directed mutation of PCR, replacing the "CCTCCC" sequence of SP3 element with the "CCTCCC" sequence of SP1 element on upstream site of-140 to construct modified promoter vector of pGL2-eNOS-repSP3. Meanwhile identification of electrophoresis and sequencing analysis were carried out to identify the vector and PCR products. Designed a human umbilical vein endothelial cell-12(HUVEC-12)'s hypoxia model that induced by Cobalt chloride (CoCl2) in vitro. The cells were transfected with normal and modified eNOS promoter vectors repsectively. Compared with another modified eNOS promoter(pGL2-eNOS-insSPl) the transcriptional activity of pGL2-eNOS-repSP3 vector was determined using a double luciferase reporter gene system from cellular lysate by the stimulation of different concentrations of Cobaltous Chloride at different time. Meanwhile detect the nitric oxide (NO) concentration in cell supernatant from cell transfected with different modified vectors of eNOS promoter. RESULTS:Identification of electrophoresis and sequencing analysis indicated that the regulatory element SP3 had already been replaeced with element SP1 at eNOS promoter's-140 site and the sequence BLAST result showed that the match degree reaches 96%. NO's concentration was decreased firstly and then increased as the concentration of COCl2 increased, showing no direct correlation between them. As the stimulation time of CoCl2 lasted, NO's concentration increased too, that showed a direct correlation between them. The transcriptional activities of both pGL2-eNOS-repSP3 and pGL2-eNOS-insSPl increased, higher than that of pGL2-eNOS, as the concentration of Cobaltous Chloride increased. The transcriptional avtivities of pGL2-eNOS-repSP3 reached maximus value when concentration of CoCl2 reached 100μmol/L. When the concentration of CoCl2 was 200μmol/L, both of the three promoter's transcriptional activity decreased. The two modified promoter' transcriptional activity increased, higher than that of pGL2-eNOS, as the stimulating time of CoCl2 lasted. There was a direct correlation between stimulation time and NO's concentration from cell supernatant of the two modifed promoter vectors but not the case of concentration of CoCl2. CONCLUSION The replacement of element SP3 with SP1 up-regulate the transcriptional activities of the modified promoter vector. There is no significant difference on transcriptional activities between the two riiodified vectors, pGL2-eNOS-repSP3 and pGL2-eNOS-insSPl by the stimulation of different concentrations of Cobaltous Chloride at different time.