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HEAT SHOCK PROTEIN 70-kDA (HSP70) IN HEPG2 CELLS EXPOSED TO COPPER AND JACKSON TOXIC MILK MOUSE MODEL OF WILSON DISEASE (WD)
JYK Yap, S Yang, EA Roberts
Division of Gastroenterology, Hepatology and Nutrition; Genetics and Genome Biology Programme, Hospital for Sick Children; University of Toronto, Toronto, Ontario
Heat Shock Proteins (HSPs), notably the inducible 70-kDa protein, Hsp70, participate in cytoprotection during cellular stress including oxidant stress. WD is a genetic disorder of hepatic handling of Cu, where hepatocellular damage involves oxidant stress. We identified Hsp70 as a member of the Cu-metalloproteome (J Proteome Res 2004;3:834).
AIM: to examine the expression of Hsp70 in the human hepatoma line HepG2 exposed to copper and in liver from the Jackson toxic milk mouse (G712D missense mutation in Atp7b gene, called “tx-j” here).
METHODS: HepG2 cells were cultured in increasing concentrations of CuCl (0-50-100-150 uM) for 24 and 48 hours; incubation with ZnCl over the same concentration range were used as control. Hsp70 protein and mRNA expression were examined by immunoblotting and semi-quantitative RTPCR, respectively. The same analyses were performed on the livers of tx-J mice, 1-6 months-old.
RESULTS: In the first 24 hours, the Hsp70 protein concentration in HepG2 cells increased with higher incubating concentrations of Cu but plateaued at 48 hours. HepG2 cells incubated in Zn for 24 hours showed no increase in Hsp70. Hsp70 mRNA expression paralleled Hsp70 protein expression. In the tx-j mouse, although Hsp70 protein concentration peaked at 3 months, the overall pattern of Hsp70 protein expression was similar in tx-j and control mice at all ages studied. Hsp70 mRNA expression in tx-j mouse was up-regulated from the first month of life onwards, whereas the control mouse experienced a gradual increase of HSP70 mRNA expression over the first 6 months.
CONCLUSION: Changes in Hsp70 expression in HepG2 cells exposed to Cu are consistent with a response to oxidant stress. In Wilsonian mice HSP70 appears to play a limited role in physiological adjustments as a result of impaired hepatic Cu-handling.