| It has been known that hypoxia increases glycolysis metabolism, to keep up with this increased glycolysis, there must be an increased uptake of the substrate, glucose. One of the proteins responsible for the uptake of glucose into the cells is Glut-1, which was the first of a family of glucose transporters to be cloned. Glut-1 is expressed in most normal tissues and is responsible for basal glucose transport. Numerous factors have been shown to regulate Glut-1 mRNA expression, including phorbol esters, oncogenes, hypoxia, growth factors and mitogens. HIF-1 is composed of HIF-1αand HIF-1β, a master regulator of oxygen homeostasis, and is playing critical roles in physiological and pathological process. HIF-1 target genes encode proteins that increase O2 delivery and mediate adaptive response to O2 deprivation, including angiogensis/vascular remodeling, erythropoiesis, glycolysis, iron transporter and cell proliferation/survival. But the changes and its mechanisms of Glut-1 protein and Glut-1 mRNA of cells in burned organisms, especially the role of HIF-1 is still not clear.Objective: To investigate the change of Glut-1 in the severe burned rats and survey the role of HIF-1 of rat cells in the changes of Glut-1 mRNA transcroption.Methods: The Wistar rats inflicted with 30% TBSA full thickness flame burn on the back were killed on 0.5h, 1h, 2h, 4h, 8h, 16h postburn and the amount of Glut-1 protein in the rat liver were determined by Western Blotting. Construct N containing the potential HIF-1 binding site was constructed by using Construct A which contains the full length of the 5'-flanking region of the rat Glut-1 gene as a template of PCR, and the PCR product was subcloned into the reporter plasmid pGL3-Promoter. Construct M was made using a two-step overlapping PCR strategy. Then the liver cell lines BRL were transfected with Construct N, Construct M or cotransfected with p(HA)HIF-1 respectively, 24 hours later, the cells were subjected to hypoxia (1%O2) to mimic the hypoxia enviroment caused by burns for 12 hours. In order to examine the effect of kinase inhibitors on the reporter gene assay, the cells were treated with SB203580(50uM, in DMSO) or U0126(20uM, in DMSO) for 1 hour before subject to hypoxia and luciferaseactivity and pSV-β-Galactosidase assay were performed.Results: ①Compared with normal control, the content of Glut-1 protein in the liver was significantly increased, as time went by ,the peak levels were observed at about 4-8h time point. ②Construct N and Construct M were successfully constructed. ③The liver cell lines was successfully transfected with Construct A and the expression of the luciferase was significantly induced by hypoxia, as compared with the control which was not subjected to hypoxia, the luciferase activity reached the peak at 12h induced by hypoxia.④Using Construct N and Construct M transfect the cell lines, hypoxia induced the luciferase activity of Construct N and Construct M. When cotransfected into the BRL with an expression vector encoding rat HIF-1α, hypoxia and the HIF-1 vector were synergistic in construct N transfected cells and luciferase activity was induced to about 16-fold, but not construct M.⑤Pretreated the cells transfected with construct N with SB203580 decrease the hypoxia-induced expression of the reporter gene about 52% compared with that in hypoxia conditions, we also found, using another inhibitor of ERK U0126 treated the cells, the hypoxia-induced expression of the reporter gene decreased about 49% compared with that in hypoxia. But pretreated the cells transfected with construct M with SB203580 or U0126 did not change the activity of luciferase. Conclusion: In this study, wild type HRE plasmid (construct N) and mutation type HRE plasmid(construct M) were successfully constructed and transfected into the rat liver cell lines, hypoxia and the HIF-1 induced the expressin of luciferase activity through the HBS of the HRE. Moreover, p42/p44, p38MAPK participate in the process of the transcription of HIF-1 target genes.
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