Induction of Adaptive Response and Enhancement of PC12 Cell Tolerance by 7-Hydroxycholesterol and 15-Deoxy-Δ12,14-Prostaglandin J2 through Up-regulation of Cellular Glutathione via Different Mechanisms

Zhi-Hua Chen,Azusa Sekine,Etsuo Niki,Yasukazu Yoshida,Noriko Noguchi,Yoshiro Saito

doi:10.1074/jbc.m600260200

Abstract

Increasing evidence suggests an adaptive response induced by reactive oxygen species and other physiologically existing oxidative stimuli. We have recently reported that a variety of lipid peroxidation products at sublethal concentrations could induce adaptive response and enhance PC12 cell tolerance, although the detailed underlying molecular mechanisms have not been clearly clarified. In the present study, we found that both 7-hydroxycholesterol (7-OHCh) and 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) at sublethal concentrations significantly increased the cellular GSH as well as the enzyme activity of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis. Depletion of cellular GSH by buthionine sulfoximine completely abolished the adaptive response. Interestingly, treatment with 15d-PGJ2 significantly increased the gene expression of both subunits of GCL in an NF-E2-related factor 2 (Nrf2)-dependent manner, whereas neither 7-OHCh induced any considerable changes on the GCL gene expression nor did the Nrf2-small interfering RNA treatment exert any appreciable effects on the GSH elevation and subsequent adaptive response induced by 7-OHCh. These results demonstrate that the adaptive response induced by both 7-OHCh and 15d-PGJ2 is mediated similarly through the up-regulation of GSH but via different mechanisms.

Highlights

Products, like other reactive oxygen species (ROS), have been considered to play an important role in cellular signal transduction
Prostaglandins of the J series (PGJs) are cyclopentenones synthesized from arachidonic acid via enzymatic conversion by cyclooxygenase and prostaglandin D2 synthase followed by nonenzymatic dehydration from prostaglandin D2 to a series of PGJs, including 15-deoxy-⌬12,14-prostaglandin J2 (15d-PGJ2) [5]
Adaptive Response Induced by 7-OHCh and 15d-PGJ2 in PC12 Cells— To explore the possible adaptive response, the cytotoxicity induced by these compounds was first investigated in PC12 cells

Summary

EXPERIMENTAL PROCEDURES

Materials—Authentic 7␣-and 7␤-OHCh were obtained from Steraloids Inc., and 15d-PGJ2 was a product from Calbiochem. Measurement of Cellular GSH Contents—Intracellular GSH contents, including both reduced and oxidized forms, were determined enzymatically by a modified 5,5Ј-dithiobis-(2-nitrobenzoic acid) assay according to the method described elsewhere [23]. The blots were blocked for 1 h at room temperature in fresh blocking buffer (0.1% Tween 20 in Tris-buffered saline, pH 7.4, containing 5% nonfat dry milk). After three washes with phosphate-buffered saline and 0.1% Tween 20, the blots were incubated with the horseradish peroxidase-conjugated secondary antibody in phosphate-buffered saline with 3% nonfat dry milk for 1 h at room temperature. The blots were again washed three times in phosphate-buffered saline and 0.1% Tween 20 buffer, and the transferred proteins were incubated with ECL solution (Amersham Biosciences) for 1 min, in accordance with the manufacturer’s instructions, and visualized on radiographic film. Values of p Ͻ 0.05 were considered to be statistically significant

RESULTS

Increased GCL Activity and Subsequent GSH Elevation Were

DISCUSSION