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. 2014 Sep 5;289(36):24944-55.
doi: 10.1074/jbc.M114.580357. Epub 2014 Jul 21.

Proteasome dysfunction activates autophagy and the Keap1-Nrf2 pathway

Shun Kageyama  1 , Yu-shin Sou  2 , Takefumi Uemura  3 , Satoshi Kametaka  3 , Tetsuya Saito  4 , Ryosuke Ishimura  4 , Tsuguka Kouno  2 , Lynn Bedford  5 , R John Mayer  5 , Myung-Shik Lee  6 , Masayuki Yamamoto  7 , Satoshi Waguri  3 , Keiji Tanaka  8 , Masaaki Komatsu  9
Affiliations

Proteasome dysfunction activates autophagy and the Keap1-Nrf2 pathway

Shun Kageyama et al. J Biol Chem. .

Abstract

The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. These pathways are interdependent, and dysfunction in either pathway causes accumulation of ubiquitin-positive aggregates, a hallmark of human pathological conditions. To elucidate in vivo compensatory action(s) against proteasomal dysfunction, we developed mice with reduced proteasome activity in their livers. The mutant mice exhibited severe liver damage, accompanied by formation of aggregates positive for ubiquitin and p62/Sqstm1, an adaptor protein for both selective autophagy and the anti-oxidative Keap1-Nrf2 pathway. These aggregates were selectively entrapped by autophagosomes, and pathological features of livers with impaired proteasome activity were exacerbated by simultaneous suppression of autophagy. In contrast, concomitant loss of p62/Sqstm1 had no apparent effect on the liver pathology though p62/Sqstm1 was indispensable for the aggregates formation. Furthermore, defective proteasome function led to transcriptional activation of the Nrf2, which served as a physiological adaptation. Our in vivo data suggest that cells contain networks of cellular defense mechanisms against defective proteostasis.

Keywords: Autophagy; Autophagy-related Protein 7 (ATG7); Nuclear Factor 2 (Erythroid-derived 2-Like Factor) (NFE2L2) (Nrf2); Proteasome; Protein Aggregation.

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Figures

FIGURE 1.
FIGURE 1.
Time course analysis of Rpt2f/f;Alb mice. A, liver homogenates were prepared from mice of the indicated genotypes at P20, P30, and P40 and subjected to immunoblotting with the indicated antibodies. Data were obtained from three independent experiments. B, proteasome activity in Rpt2f/f;Alb livers at P20, P30, and P40. C, growth curve of Rpt2f/f (control) and Rpt2f/f;Alb mice. Data are means ± S.E. of Rpt2f/f (n = 33) and Rpt2f/f;Alb (n = 24) mice. ***, p < 0.001. D, ratio of liver weight to body weight of Rpt2f/f (control) and Rpt2f/f;Alb mice at P30. Data are means ± S.E. of Rpt2f/f (n = 5) and Rpt2f/f;Alb (n = 8) mice. E, H&E staining of livers of indicated genotypes at P30. Mitotic cells or abnormal mitosis (arrowheads) were often observed in Rpt2-deficient hepatocytes. CV, central vein; P, portal triad. Bar, 100 μm. F, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured. Data are means ± S.E. of Rpt2f/f (n = 3) and Rpt2f/f;Alb (n = 5) mice. ***, p < 0.001. IU/L, international units/liter. AFU, arbitrary fluorescent unit.
FIGURE 2.
FIGURE 2.
Characterization of ubiquitinated aggregates in Rpt2f/f;Alb livers. A, liver homogenates were prepared from mice of the indicated genotypes at P30. Total, soluble (Sol.), and insoluble (Insol.) fractions were subjected to immunoblotting with the indicated antibodies. Data were obtained from three independent experiments. B, liver cryosections from mice of the indicated genotypes at P30 were double-immunostained with p62 and ubiquitin antibodies. A portion of each image is magnified and shown in the inset. Arrows indicate large pleomorphic aggregated structures. Merged images are shown at the right column (red, p62; green, ubiquitin). Bars, 20 μm. C, electron micrographs of hepatocytes of the indicated genotypes. The boxed regions in a′, b′, and d′ are enlarged and shown in b′, c′, and e′, respectively. Arrowheads indicate aggregated structures. Bars, a′, 1 μm; b′ and c′, 0.5 μm; d′, 0.1 μm. D, immunoelectron micrographs showing double labeling of ubiquitin (12-nm colloidal gold particles) and p62 (6-nm colloidal gold particles) in hepatocytes of Rpt2f/f;Alb mice at P30. The boxed region is enlarged and shown at the right. Bars, 0.2 μm.
FIGURE 3.
FIGURE 3.
Autophagic degradation of ubiquitin-positive aggregates accumulated due to impaired proteasome activity. A, mice of the indicated genotypes were subjected to intraperitoneal injection of leupeptin at P30. One hour after the injection, liver homogenates were prepared and subjected to immunoblotting with the indicated antibodies. Data were obtained from three independent experiments. A graph indicates quantitative densitometry of immunoblotting data (n = 3) and the ratios of insoluble (Insol.) ubiquitinated proteins relative to that of dimethyl sulfoxide (DMSO)-treated control mice. B, liver cryosections from mice of the indicated genotypes treated as described in A were double-immunostained with LC3 (red in the merged image) and ubiquitin (green in the merged image) antibodies. The boxed region is magnified and shown in the inset. Bars, 10 μm. C, electron micrographs of hepatocytes from mice of the indicated genotypes, treated as described in A. Arrowheads, aggregate-like amorphous structures. G, glycogen granules. Bar, 0.2 μm. Sol., soluble.
FIGURE 4.
FIGURE 4.
Exacerbation of pathology in Rpt2f/f;Alb liver by concomitant loss of Atg7. A, liver homogenates were prepared from mice of the indicated genotypes at P30. Total, soluble (Sol.), and insoluble (Insol.) fractions were subjected to immunoblotting with the indicated antibodies. Data were obtained from three independent experiments. B, liver cryosections of Rpt2f/f;Atg7f/f;Alb mice were double-immunostained with p62 and ubiquitin antibodies. A portion of each image was magnified and shown in the inset. Arrows indicate large pleomorphic aggregated structures. Merged images are shown in the right column (red, p62; green, ubiquitin). Bars, 20 μm. C, electron micrographs of Rpt2f/f;Atg7f/f;Alb hepatocytes. Arrowheads indicate aggregated structures. Bar, 1 μm. D, growth curves of mice of the indicated genotypes. Data are means ± S.E. of control (n = 35), Atg7f/f;Alb (n = 22), Rpt2f/f;Alb (n = 10), and Rpt2f/f;Atg7f/f;Alb (n = 17) mice. **, p < 0.01; ***, p < 0.001. E, ratio of liver weight to body weight of mice of the indicated genotype at P30. Data are means ± S.E. of control (n = 19), Atg7f/f;Alb (n = 13), Rpt2f/f;Alb (n = 10), and Rpt2f/f;Atg7f/f;Alb (n = 8) mice. *, p < 0.05; ***, p < 0.001. F, H&E staining of livers of the indicated genotypes at P30. P, portal triad; CV, central vein. Bar, 100 μm. G, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured. Data are means ± S.E. of control (n = 17), Atg7f/f;Alb (n = 13), Rpt2f/f;Alb (n = 8), and Rpt2f/f;Atg7f/f;Alb (n = 16) mice. **, p < 0.01; ***, p < 0.001. IU/L, international units/liter.
FIGURE 5.
FIGURE 5.
Feedback activation of the Keap1-Nrf2 pathway serves as a physiological adaptation to impaired proteasome function. A, total lysates, detergent-soluble (Sol.) and -insoluble (Insol.) fractions, and nuclear fractions from livers of the indicated genotypes were subjected to immunoblotting with the indicated antibodies. Data were obtained from three independent experiments. B, liver cryosections from mice of the indicated genotypes were double-immunostained with antibodies against p62 and Keap1 antibodies. Arrows indicate large pleomorphic aggregated structures. Merged images are shown in the right column of each panel (red, p62; green, Keap1). Bars, 20 μm. C, total RNAs were prepared from livers of the indicated genotypes. Values were normalized to the amount of mRNA in the livers of control mice. Data are means ± S.E. of control (n = 14), Nrf2−/− (n = 10), Rpt2f/f;Alb (n = 5), and Rpt2f/f;Alb;Nrf2−/− (n = 7) mice. *, p < 0.05. D, growth curves of mice of the indicated genotypes. Data are means ± S.E. of control (n = 15), Nrf2−/− (n = 13), Rpt2f/f;Alb (n = 6), and Rpt2f/f;Alb;Nrf2−/− (n = 6) mice. *, p < 0.05; ***, p < 0.001. E, H&E staining of livers of the indicated genotypes at P30. P, portal triad; CV, central vein. Bar, 100 μm. F, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured. Data are means ± S.E. of control (n = 13), Nrf2−/− (n = 11), Rpt2f/f;Alb (n = 6), and Rpt2f/f;Alb;Nrf2−/− (n = 7) mice. **, p < 0.01; ***, p < 0.001. IU/L, international units/liter.
FIGURE 6.
FIGURE 6.
Role of p62 in formation of aggregate-containing structures and Nrf2 activation in Rpt2f/f;Alb hepatocytes. A, total lysates, detergent-soluble (Sol.) and -insoluble (Insol.) fractions, and nuclear fractions from livers of the indicated genotypes were subjected to immunoblotting with the indicated antibodies. Data were obtained from three independent experiments. B, liver cryosections from mice of the indicated genotypes were double-immunostained with p62 and ubiquitin antibodies. A portion of each image is magnified and shown in the inset. Arrows indicate large pleomorphic aggregated structures. Merged images are shown in the right column (red, p62; green, ubiquitin). Bars, 20 μm. The graph shows the average number (± S.E.) of ubiquitin-positive large aggregates counted in an area of 210 × 210 μm in liver sections from three animals for each genotype (n = 30). C, electron micrograph of Rpt2f/f;p62f/f;Alb hepatocytes. The boxed regions in a′ and b′ are enlarged and shown in b′ and c′, respectively. Arrowheads indicate aggregated structures. Bars, a′, 1 μm; b′, 0.5 μm; c′, 0.1 μm. D, total RNAs were prepared from livers of the indicated genotypes. Values were normalized to the amount of mRNA in the livers of control mice. Data are means ± S.E. of Rpt2f/f (n = 4), Rpt2f/f;Alb (n = 8), p62f/f (n = 13), p62f/f;Alb (n = 10), Rpt2f/f;p62f/f (n = 11), and Rpt2f/f;p62f/f;Alb (n = 12) mice. *, p < 0.05; **, p < 0.01.
FIGURE 7.
FIGURE 7.
Pathology in Rpt2f/f;Alb liver by concomitant loss of p62. A, growth curves of mice of the indicated genotypes. Data are means ± S.E. of control (n = 44), p62f/f;Alb (n = 9), Rpt2f/f;Alb (n = 6), and Rpt2f/f;p62f/f;Alb (n = 15) mice. ***, p < 0.001. B, H&E staining of livers of the indicated genotypes at P30. P, portal triad; CV, central vein. Bar, 100 μm. C, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured. Data are means ± S.E. of control (n = 31), p62f/f;Alb (n = 5), Rpt2f/f;Alb (n = 5), and Rpt2f/f;p62f/f;Alb (n = 12) mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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