Regulatory role of Epo3'-enhancer in cellular hypoxic response
- Author:
Xianrong JIN
- Publication Type:Journal Article
- From:
Chinese Journal of Pathophysiology
2001;17(8):770-771
- CountryChina
- Language:Chinese
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Abstract:
Wild type fragment of erythropoietin(Epo)3'-enhancer (W18) and its mutant type fragment (M18) were synthesized. Primary cultures of endothelial cells of different sources (ECs) and human umbilical venous endothelial cell line (HUVEC) as well as the primary culture of pulmonary artery smooth muscle cells (PASMC) were transfected with either W18 or M18. RT-PCR was performed to detect mRNA and investigate the effect of Epo3'-enhancer fragment on the hypoxia- induced gene expression in ECs. Electrophoretic mobility shift assay(EMSA) was used to test DNA-binding activity of extracted nuclear protein. ［3H］-TdR incorporation, MTT test and flow-cytometry were used to determine the proliferation of PASMC and the effect of Epo3'-enhancer fragment on it. The results showed as follows:(1)The OD value of COX-2 mRNA expressed in hypoxic rat aortic EC was 2.34±0.32, the OD values of COX-2 and VEGF mRNAs expressed in hypoxic EC of pulmonary microvasculature were 1.78±0.21 and 4.71±0.52, the OD value of TXS and ET-1 mRNAs in hypoxic HUVEC were 13.01±4.27 and 1.01±0.05, they were all higher than their counterpart normoxic group (P＜0.05). If the cells were pretransfected with W18, the OD values in these hypoxic groups became 1.28±0.25,0.77±0.09, 2.29±0.41, 4.88±1.05. and 0.51±0.15, respectively, just as low as those of the relevant normoxic groups; (2)The conditioned medium of hypoxic pulmonary artery endothelial cell (PAEC) caused proliferation of PASMC, the cpm values of ［3H］-TdR incorporation was 917.00±527.11, higher than that of normoxic control (P＜0.05). In addition, it was found by using flow-cytometry an increase in the percentage of cells of phases S and G2/M in PASMC incubated in hypoxic EC conditioned medium in comparison with normoxia group and group pretransfected with W18; (3)Hypoxia could induce proliferation of PASMC directly, transfection of W18 could decrease its effect. Their cpm values were 829.50±228.10 and 497.00±52.45, respectively(P＜0.05).The results of MTT test was similar; (4)The inducer of HIF-1, CoCl2 could increase the expression of COX-2 and TXS mRNA in HUVEC, which could also be inhibited by W18 but not by M18;(5)The HIF-1 DNA binding activity was found in hypoxic HUVEC in EMSA with 32 P labeled W18, but not found with 32 P M18. These results suggest that there might be a common pathway in hypoxic responses of different cells, i.e. regulating the transcription of genes by binding of HIF-1 to a sequence similar to Epo3'-enhancer.
