Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway

Abstract

Proteasomes degrade damaged proteins formed during oxidative stress, thereby promoting cell survival. Neurodegenerative and other age-related disorders are associated with reduced proteasome activity. We show herein that expression of most subunits of 20S and 19S proteasomes, which collectively assemble the 26S proteasome, was enhanced up to threefold in livers of mice following treatment with dithiolethiones, which act as indirect antioxidants. Subunit protein levels and proteasome activity were coordinately increased. No induction was seen in mice where the transcription factor Nrf2 was disrupted. Promoter activity of the PSMB5 subunit of the 20S proteasome increased with either Nrf2 overexpression or treatment with antioxidants in mouse embryonic fibroblasts. Tandem antioxidant response elements in the proximal promoter of PSMB5 that controlled these responses were identified. We propose that induction of the 26S proteasome through the Nrf2 pathway represents an important indirect action of these antioxidants that can contribute to their protective effects against chronic diseases.

FIG. 1.

Microarray summary of proteasome subunits induced in mouse liver by D3T. (A) Proteasomes degrade oxidatively damaged proteins as well as ubiquitin-marked proteins. (B) Subunit components of the 19S and 20S proteasomes. Subunits in boldface italics are genes elevated by D3T in livers of wild-type, but not nrf2-disrupted, mice. Subunits in boldface only are genes induced by D3T in both genotypes.

FIG. 2.

Induction of 26S transcript and protein levels of subunits by D3T in livers of wild-type mice. (A) mRNA levels for 26S proteasome subunits. Hepatic transcript levels of each subunit in wild-type and nrf2-disrupted mice 24 h after treatment with D3T were measured by RT-PCR analysis. Each sample was prepared from three pooled livers. (B) Induction of protein levels for 26S proteasome subunits PSMA1, PMB5, and PSMC1 in wild-type mouse liver by D3T. Immunoblot analyses were performed using subunit-specific antibodies. Each sample was prepared from three pooled livers, and three similar immunoblots were obtained.

FIG. 2.

Induction of 26S transcript and protein levels of subunits by D3T in livers of wild-type mice. (A) mRNA levels for 26S proteasome subunits. Hepatic transcript levels of each subunit in wild-type and nrf2-disrupted mice 24 h after treatment with D3T were measured by RT-PCR analysis. Each sample was prepared from three pooled livers. (B) Induction of protein levels for 26S proteasome subunits PSMA1, PMB5, and PSMC1 in wild-type mouse liver by D3T. Immunoblot analyses were performed using subunit-specific antibodies. Each sample was prepared from three pooled livers, and three similar immunoblots were obtained.

FIG. 3.

Enhanced peptidase activities of proteasomes in wild-type mouse liver following treatment with D3T. Chemoptypsin-like (Suc-LLVY) (A), postglutamic (Z-LLE) (B), and trypsin-like (Z-ARR) (C) peptidase activities in liver homogenates prepared from wild-type and nrf2-disrupted mice were measured. Values are means ± standard errors from three experiments. a, P < 0.05 compared with vehicle-treated control group.

FIG. 4.

Effect of antioxidants on the level of PSMB5 transcripts in murine embryonic fibroblasts. Transcript levels for PSMB5 were measured following treatment with antioxidants D3T (30 μM), sulforaphane (Sul; 10 μM), butylhydroxytoluene (BHT; 100 μM), and ethoxyquin (EQ; 80 μM) for 18 h in fibroblasts from wild-type and nrf2-disrupted mice. The histogram depicts the means ± standard errors from three separate experiments. a, P < 0.05 compared with vehicle-treated controls.

FIG. 5.

The promoter of PSMB5 is activated by sulforaphane treatment or Nrf2 overexpression. Murine PSMB5 promoter constructs (A) and luciferase activities derived from these truncated promoters following treatment with sulforaphane (10 μM) or cotransfection of an Nrf2 expression plasmid (B) are shown. Two tandem AREs in an inverted direction were identified 341 and 52 bp upstream of the PSMB5 gene coding region. Arrows indicate the orientation of these putative AREs. a, P < 0.05 compared with blank plasmid-transfected, vehicle-treated control. SV40, simian virus 40.

FIG. 5.

The promoter of PSMB5 is activated by sulforaphane treatment or Nrf2 overexpression. Murine PSMB5 promoter constructs (A) and luciferase activities derived from these truncated promoters following treatment with sulforaphane (10 μM) or cotransfection of an Nrf2 expression plasmid (B) are shown. Two tandem AREs in an inverted direction were identified 341 and 52 bp upstream of the PSMB5 gene coding region. Arrows indicate the orientation of these putative AREs. a, P < 0.05 compared with blank plasmid-transfected, vehicle-treated control. SV40, simian virus 40.

FIG. 6.

The promoter of PSMB5 is regulated by the Nrf2-ARE pathway. (A) Luciferase activity driven by the proximal PSMB5 promoter (−1.1kb-luc) following treatment with antioxidants in wild-type and nrf2-disrupted cells. BHT, butylhydroxytoluene; EQ, ethoxyquin. (B) Elevated basal activity of the proximal PSMB5 promoter (−1.1kb-luc) in keap1-disrupted cells. Overexpression of MafK inhibited promoter activation by Nrf2. a, P < 0.05 compared with blank plasmid-transfected, vehicle-treated control; b, P < 0.05 compared with pcDNA3-Nrf2-transfected group. WT, wild type.

FIG. 7.

Response of mutated PSMB5 promoter to sulforaphane (Sul) treatment and Nrf2 overexpression in wild-type cells. AREs located at −341 and −52 were mutated, and singly and doubly mutated promoters were generated from the proximal promoter construct (−1.1kb-luc). Values are means ± standard errors from three experiments. a, P < 0.05 compared with blank plasmid-transfected, vehicle-treated control.

FIG. 8.

Binding of Nrf2 to PSMB5 promoter in intact cells. (A) Chromatin immunoprecipitants with no antibody (Ab), immunoglobulin G (IgG), GATA-1, or Nrf2 were used for PCR amplification of each promoter. ARE-containing promoter regions from PSMB5 and the GSTA1 promoter were detected in Nrf2 immunoprecipitants obtained from sulforaphane-treated wild-type cells. (B) Enhanced binding of Nrf2 to the PSMB5 promoter following sulforaphane (Sul) treatment compared to that in vehicle (V)-treated cells. Promoter regions of GSTA1 and PSMB5 genes were detected in Nrf2-immunoprecipitants from keap1-disrupted cells, but not nrf2-disrupted cells. WT, wild type.