Sp1 Up-regulates The Expression Of ETS2 Gene And Its Molecular Mechanism

Objective: The ETS2 gene is located on human chromosome21q22.3,which plays an important role in essential biological processes such as growth,transformation,apoptosis,differentiation,and organogenesis.Increased dosage of Ets2 has led to skeletal abnormalities,apoptosis of neurons,transactive of ?-Amyloid precursor protein(?-APP)gene,suppress solid tumour in Down syndrome suggesting important role for Ets2 in the pathology of Down syndrome.In the present study,we aimed to uncover the transcriptional regulation of the ETS2 gene.Materials and methods: primers design,molecular cloning,plasmid construction,cell culture and transfection,dual luciferase assay,nucleoprotein extract,electrophoretic mobility shift assay,Western Blot.Results: 1.The 1191 bp 5' flanking region of ETS2 gene was cloned into the promoter-lacking plasmid vector pGL4.10 to generate pETS2-A(-1058 to +132),and a series of deletion within p ETS2 –A was generated.The luciferase assays of these deletion plasmids were performed,some positive regulatory elements were found.2.Transcritional factor binding site analysis revealed that the human ETS2 gene promoter +48 to +132 contained several Sp1 binding sites.3.EMSA results confirmed that the human ETS2 promoter +48 to +70bp contains a Sp1 binding site.When the Sp1 site was mutant,the pETS2-A Mut promoter activity was down to 40% compared to pETS2-A.4.The plasmid pETS2-A was co-transfected with pCGN-Sp1 into HEK293 and SH-SY5 Y cells.The results showed that Sp1 overexpression significantly increased ETS2 promoter activity to 2.37-fold(p<0.05)and 2.5-fold(p<0.05)respectively.5.Sp1 overexpression in HEK293 and SH-SY5 Y cells increased ETS2 mRNA level to 1.43 fold(p<0.05)and 1.39 fold(p<0.05)respectively.6.Sp1 overexpression in HEK293 and SH-SY5 Y cells significantly increased ETS2 protein level to 2.74-fold(p<0.0001)and 1.96-fold(.p<0.001)In addition,knockdown of Sp1 protein level in HeLa cell decreased ETS2 protein level to 1.6-fold(p<0.05)Conclusion: The 5' flanking regeion-1058 to +132 of ETS2 gene has a strong promoter activity which contains functional Sp1 binding site.And the ranscription factor Sp1 can bind to ETS2 gene confirmed by EMSA experiment.Cotansfecting ETS2 reporter gene with Sp1 up-regulates the human ETS2 gene promoter activity.Importantly,Overexpression of Sp1 not only significantly increased ETS2 gene mRNA level but also protein level.In turn,knockdown of Sp1 protein level in HeLa cell decreased ETS2 protein level.In conclusion,ETS2 gene expression can be up-regulated by Sp1.Objective: To investigate the effects of carnosine(CAR)on oxidative stress and inflammatory factors in mice with liver injury induced by carbon tetrachloride(CCl4).Materials and methods: Thirty ICR mice were randomly divided into three groups: control group,CCl4 group and CAR group.Except for control group,CCl4 group and CAR group were exposed with the 0.2%(v/v)CCl4/peanut oil solution(10 m L/kg)by intragastric administration.Eight hours later,CAR group was treated with 300 mg/kg carnosine for continuous seven days.At the eighth day,the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),albumin(ALB)and glutamyltranspeptidase(GGT)in the serum were detected by biochemistry analyzer;the activity of superoxide dismutase(SOD)and the concentration of malonaldehyde(MAD)and glutathion(GSH)were measured by ELISA;the expression levels of cyclooxygenase-2(COX-2)and heineoxygenase-1(HO-1)were analyzed by Western blot and the m RNA levels of tumor necrosis factor ?(TNF-?),interleukin1?,(IL-1?)and interleukin 10(IL-10) were detected by RT-PCR.Results: Compared with control group,the levels of ALT,AST and GGT(t=5.776,P =0.000;t=7.443,P =0.000;t=4.548,P=0.000)were significantly down-regulated and the ALB(t=6.061,P =0.000)was upregulated in the serum;the biomarkers of oxidative stress,SOD and GSH were increased(t=6.818,P =0.000;t=5.739,P =0.000),MDA was decreased(t=6.526,P =0.000);The expression level of the enzyme of oxidation COX-2(t=5.754,P =0.001)was reduced and the enzyme of oxygenation HO-1(t=2.367,P =0.001)was persistently raised;the m RNA levels of the inflammatory factors TNF-?and IL-1?(t=7.025,P =0.000;t=11.963,P =0.000)were remarkably decreased and the IL-10(t=6.074,P =0.000)was obviously up-regulated in CAR group after carnosine treatment.Conclusion: Carnosine could improve and protect chemical liver injury by attenuating the state of oxidative stress and affecting the inflammatory reaction.