[Hypoxia induced by CoCl2 influencing the expression and the activity of matrix metalloproteinase-2 in rat hepatic stellate cells].
OBJECTIVE
To investigate the effects of hypoxia induced by cobalt chloride on the expression and the activity of matrix metalloproteinase-2 (MMP-2) in rat hepatic stellate cells (HSC-T6) and to clarify the possible mechanisms.


METHODS
HSC-T6 cell line was grown in Dulbecco's modified Eagle medium with 10% fetal calf serum at 37 degrees C and 5% CO2. When reaching confluence, the cells were incubated with serum-free medium in the presence of cobalt chloride (0, 50, 100, 200 micromol/L) for six hours, and then the supernatant and the cells were harvested. The expression of the MMP-2 mRNA and HIF-1alpha protein in HSC-T6 cells was detected using RT-PCR and Western blot respectively. The activity of the MMP-2 in the supernatant was detected by zymography. The binding reaction between HIF-1a protein and MMP-2 gene sequence was investigated by electrophoresis mobility shift assay.


RESULTS
When the concentration of CoCl2 increased from 0 micromol/L to 200 micromol/L, the expressions of MMP-2 mRNA (the rate of light density) were increased from 0.53+0.12 to 1.57+0.11 and the differences among these four groups were significant (F=34.21). The activity of MMP-2 (the value of light density*band area) decreased gradually from 84.49+5.38 to 53.70+3.42, and the differences among these four groups were also significant (F=29.54). The expressions of HIF-1a were increased gradually with the increase of the CoCl2 concentration. The shift band in the lane of the nuclear protein extraction and the MMP-2 probe containing hypoxia response element showed delays when compared with the lane of the sole probe, and the binding was partially abolished when competing sense oligonucleotides were used.


CONCLUSIONS
Our results suggest that chemical hypoxia can up-regulate the expression of MMP-2 mRNA and decrease the activity of the enzyme. HIF-1alpha may play a part in the regulation of MMP-2 transcription under hypoxic conditions.
Fan
Ren-hua
RH
Department of Pathology, School of Basic Medical Sciences, Southeast University, Nanjing 210009, China.
Chen
Ping-sheng
PS
Zhao
Di
D
Zhang
Wan-dong
WD
D000818:Animals; D015687:Cell Hypoxia; D002460:Cell Line; D003035:Cobalt / Q000494:pharmacology; D055166:Hepatic Stellate Cells / Q000201:enzymology*; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D003035:Cobalt; D020778:Matrix Metalloproteinase 2; C522363:Mmp2 protein, rat; C018021:cobaltous chloride