Enhancement of heme oxygenase-1 synthesis by glutathione depletion in Chinese hamster ovary cells.

Saunders EL, Maines MD, Meredith MJ, Freeman ML
Arch Biochem Biophys. 1991 288 (2): 368-73

PMID: 1898036 · DOI:10.1016/0003-9861(91)90208-z

Chinese hamster ovary cells cultured in vitro were used to assess the role of glutathione metabolism in the induction of the 32-kDa stress protein. Enhanced synthesis of the 32-kDa protein was observed after cells were incubated with CdCl2 or diethylmaleate and protein was subjected to SDS-PAGE followed by fluorography. Concomitantly, in both cell preparations an increase in heme oxygenase activity was observed. Proteins from CdCl2- and diethylmaleate-treated cells were subjected to Western blotting and protein crossreacting with either rabbit antibody to rat liver heme oxygenase-1 (32,000 Mr) or rat testis heme oxygenase-2 (36,000 Mr) quantitated. The analysis indicated that the CdCl2 treatment increased the intensity of the HO-1 band 5.5-fold while the diethylmaleate treatment increased it three-fold relative to control. Neither treatment affected the intensity of HO-2 antibody binding. Incubation of cells with buthionine sulfoximine, under conditions which resulted in greater than or equal to 90% of the intracellular glutathione being depleted, enhanced synthesis of a 32-kDa protein when assayed by SDS-PAGE. This protein exhibited a Mr similar to the 32-kDa protein induced by either CdCl2 or diethylmaleate treatment. Proteins from buthionine sulfoximine and diethylmaleate-treated cells were mixed together and subjected to 2D PAGE. The resulting fluorograph demonstrated that both treatments produced identical patterns. In contrast, incubation of cells in diamide, a thiol oxidizing compound, resulted in enhanced synthesis of the 110-, 90-, and 73-kDa heat shock proteins but not the 32-kDa protein. The data presented have shown that depletion of glutathione by two independent methods, conjugation and inhibition of synthesis, enhances the synthesis of a 32-kDa protein identified as heme oxygenase-1; oxidation of glutathione, on the other hand did not. We interpret this to indicate that glutathione depletion rather than conjugation or oxidation represents one pathway for induction of heme oxygenase-1.

MeSH Terms (20)

Animals Antimetabolites Blotting, Western Buthionine Sulfoximine Cadmium Cadmium Chloride Cell Line Cricetinae Cricetulus Electrophoresis, Gel, Two-Dimensional Electrophoresis, Polyacrylamide Gel Female Glutathione Heat-Shock Proteins Heme Oxygenase (Decyclizing) Kinetics Maleates Methionine Sulfoximine Molecular Weight Ovary

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