Age-related decrease in proteasome expression contributes to defective nuclear factor-kappaB activation during hepatic ischemia/reperfusion

Abstract

Hepatic ischemia/reperfusion (I/R) leads to liver injury and dysfunction through the initiation of a biphasic inflammatory response that is regulated by the transcription factor nuclear factor kappaB (NF-kappaB). We have previously shown that there is an age-dependent difference in the injury response to hepatic I/R in mice that correlates with divergent activation of NF-kappaB such that young mice have greater NF-kappaB activation, but less injury than old mice. In this study, we investigated the mechanism by which age alters the activation of NF-kappaB in the liver during I/R. Young (4-5 weeks) and old (12-14 months) mice underwent partial hepatic I/R. Livers were obtained for RNA microarray analysis and protein expression assays. Using microarray analysis, we identified age-dependent differences in the expression of genes related to protein ubiquitinylation and the proteasome. In old mice, genes that are involved in the ubiquitin-proteasome pathway were significantly down-regulated during I/R. Consistent with these findings, expression of a critical proteasome subunit, non-adenosine triphosphatase 4 (PSMD4), was reduced in old mice. Expression of the NF-kappaB inhibitory protein, IkappaB alpha, was increased in old mice and was greatly phosphorylated and ubiquitinylated. The data provide strong evidence that the age-related defect in hepatic NF-kappaB signaling during I/R is a result of decreased expression of PSMD4, a proteasome subunit responsible for recognition and recruitment of ubiquitinylated substrates to the proteasome. It appears that decreased PSMD4 expression prevents recruitment of phosphorylated and ubiquitinylated IkappaB alpha to the proteasome, resulting in a defect in NF-kappaB activation.

Figure 1

Ingenuity pathway analysis identified a network of genes down regulated in response to 60 minutes of ischemia in aged mouse liver. The network is displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). Different shapes of nodes represent the functional class of the gene product. Edges describe the nature of the relationship between the nodes. A total of 17 differentially expressed focus genes involved in the proteasome pathway are identified: proteasome subunit, alpha type (PSMA) 1, 2, 4, 5, and 7; proteasome 26S subunit, ATPase (PSMC) 1,2,4,5, and 6; proteasome 26S subunit, non-ATPase (PSMD) 2, 4, 6, 7, 11, and 12; and phosducin-like (PDCL).

Figure 2

Ingenuity pathway analysis identified two networks of genes down regulated in response to 90 minutes of ischemia in aged mouse liver. (A) Network 1 shows 6 differentially expressed focus genes involved in the proteasome ubiquitin pathway which include proteasome subunit, alpha type (PSMA) 1, 4, 5, and 7; cytoplasmic FMR1 interacting protein 1 (CYFIP1); and ring finger protein 14 (RNF14). (B) Network 2 classified 14 differentially expressed focus genes involved in the proteasome ubiquitinylation pathway. Proteasome 26S subunit, non- ATPase (PSMD) 2, 4, 7, 10, 11, 12 and 14; N-myc (and STAT) interactor (NMI); histidyl-tRNA synthetase (HARS); proteasome subunit, alpha type 2 (PSMA2); proteasome 26S subunit, non-ATPase (PSMD) 3 and 6; eukaryotic translation initiation factor 3, subunit E (EIF3S6); and COP9 (constitutive photomorphogenic) homolog, subunit 6 (Arabidopsis thaliana) (COPS6).

Figure 2

Ingenuity pathway analysis identified two networks of genes down regulated in response to 90 minutes of ischemia in aged mouse liver. (A) Network 1 shows 6 differentially expressed focus genes involved in the proteasome ubiquitin pathway which include proteasome subunit, alpha type (PSMA) 1, 4, 5, and 7; cytoplasmic FMR1 interacting protein 1 (CYFIP1); and ring finger protein 14 (RNF14). (B) Network 2 classified 14 differentially expressed focus genes involved in the proteasome ubiquitinylation pathway. Proteasome 26S subunit, non- ATPase (PSMD) 2, 4, 7, 10, 11, 12 and 14; N-myc (and STAT) interactor (NMI); histidyl-tRNA synthetase (HARS); proteasome subunit, alpha type 2 (PSMA2); proteasome 26S subunit, non-ATPase (PSMD) 3 and 6; eukaryotic translation initiation factor 3, subunit E (EIF3S6); and COP9 (constitutive photomorphogenic) homolog, subunit 6 (Arabidopsis thaliana) (COPS6).

Figure 3

Ingenuity pathway analysis shows a network of 17 decreased focus genes involved in the proteasome pathway in old mice after 90 minutes of ischemia followed by 1 hour of reperfusion, which include proteasome subunit, alpha type (PSMA) 2, 4, and 7; proteasome 26S subunit, ATPase (PSMC) 1,3, and 6; proteasome 26S subunit, non-ATPase (PSMD) 4, 7, 10, 11, and 12; eukaryotic translation initiation factor 3, subunit E (EIF3S6); COP9 homolog, subunit (COPS) 2, 4 and 6; nuclear receptor subfamily 3, group C, member 1 (NR3C1); and glutamate-ammonia ligase (glutamine synthetase) (GLUL).

Figure 4

Effects of age on protein expression of total (upper panel) and phosphorylated (lower panel) IκBα. Cytoplasmic fractions of whole liver tissue from from young (4-5 weeks) and old (12-14 months) mice undergoing sham surgery, 90 minutes of ischemia (90/0), or ischemia and 1 hour of reperfusion (90/1) were immunoblotted for total IκBα or phosphorylated IκBα (p-IκBα).

Figure 5

Effects of age on polyubiquitinylation of IκBα during hepatic I/R injury. Cytoplasmic fractions from young (4-5 weeks) and old (12-14 months) mice undergoing sham surgery, 90 minutes of ischemia (90/0), or ischemia and 1 hour of reperfusion (90/1) were immunoprecipited (IP) with anti-IκBα followed by immunoblotting (IB) with anti-polyubiquitin.

Figure 6

Effects of age on proteasome proteolytic activity. Proteasome activity was determined in liver extracts from young (4-5 weeks) and old (12-14 months) mice undergoing sham surgery, 90 minutes of ischemia (90/0), or ischemia and 1 hour of reperfusion (90/1). Data are expressed as mean ± SEM with n=3 per group.

Figure 7

Effects of age on protein expression of proteasome subunits. (A) Protein expression of PSMA5 (upper panel) and PSMD4 (lower panel) in cytoplasmic fractions of whole liver tissue from young (4-5 weeks) and old (12-14 months) mice undergoing sham surgery, 90 minutes of ischemia (90/0), or ischemia and 1 hour of reperfusion (90/1). PSMD4 expression was quantitated by image analysis. Data represent mean relative intensity units (RIU) ± SEM with n=3 per group. (B) Protein expression of PSMD4 in isolated Kupffer cells (KC) and hepatocytes (Hep) from young and old mice. Extracts from whole liver (WL) from young mice served as a control.

Figure 8

Effect of PSMD4 knockdown on TNFα-induced IκBα degradation in hepatocytes. (A) Transfection of AML-12 cells with non-specific (NS) or PSMD4 siRNA. (B) Effects of TNFα (10 ng/ml for 15 minutes) on untransfected AML-12 cells or cells transfected with NS or PSMD4 siRNA.