Enhancement of heme oxygenase-1 synthesis by glutathione depletion in Chinese hamster ovary cells.
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.
Saunders
E L
EL
Vanderbilt Center for Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Maines
M D
MD
Meredith
M J
MJ
Freeman
M L
ML
D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D015153:Blotting, Western; D019328:Buthionine Sulfoximine; D002104:Cadmium / Q000494:pharmacology; D019256:Cadmium Chloride; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008298:Maleates / Q000494:pharmacology; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D008970:Molecular Weight; D010053:Ovary
D000963:Antimetabolites; D006360:Heat-Shock Proteins; D008298:Maleates; D002104:Cadmium; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D006419:Heme Oxygenase (Decyclizing); C014476:diethyl maleate; D005978:Glutathione; D019256:Cadmium Chloride