Role and Regulation of p65/β-Catenin Association During Liver Injury and Regeneration: A "Complex" Relationship

Abstract

An important role for β-catenin in regulating p65 (a subunit of NF-κB) during acute liver injury has recently been elucidated through use of conditional β-catenin knockout mice, which show protection from apoptosis through increased activation of p65. Thus, we hypothesized that the p65/β-catenin complex may play a role in regulating processes such as cell proliferation during liver regeneration. We show through in vitro and in vivo studies that the p65/β-catenin complex is regulated through the TNF-α pathway and not through Wnt signaling. However, this complex is unchanged after partial hepatectomy (PH), despite increased p65 and β-catenin nuclear translocation as well as cyclin D1 activation. We demonstrate through both in vitro silencing experiments and chromatin immunoprecipitation after PH that β-catenin, and not p65, regulates cyclin D1 expression. Conversely, using reporter mice we show p65 is activated exclusively in the nonparenchymal (NPC) compartment during liver regeneration. Furthermore, stimulation of macrophages by TNF-α induces activation of NF-κB and subsequent secretion of Wnts essential for β-catenin activation in hepatocytes. Thus, we show that β-catenin and p65 are activated in separate cellular compartments during liver regeneration, with p65 activity in NPCs contributing to the activation of hepatocyte β-catenin, cyclin D1 expression, and subsequent proliferation.

Figure 1

Expression of β-catenin and p65 change over time in wild-type (WT) livers after d-galactosamine (GalN)/lipopolysaccharide (LPS). (A) WB shows that cleavage of β-catenin occurs as early as 3 h after GalN/LPS treatment in WT livers, with near-complete degradation occurring by 6 h. Expression of some β-catenin targets such as GS and Axin2 remains unchanged over time, while others such as cyclin D1 decrease with increasing β-catenin degradation. (B) Expression of p65 also decreases in WTs after GalN/LPS treatment and is absent by 6 h; however, in KO1, p65 expression persists after GalN/LPS treatment and increases at 6 h.

Figure 2

LPR5/6 KO mice are susceptible to GalN/LPS-induced liver injury. (A) Immunoprecipitation (IP) shows that p65 and β-catenin associate strongly in both WT and LRP5/6 KO (KO2) livers at baseline. (B) Kaplan–Meier analysis of WT and KO2 mice shows no difference in survival after GalN/LPS (n ≥ 3). (C) Hematoxylin and eosin (H&E) (top) shows massive inflammation, necrosis, and hemorrhage in both WT and KO2; terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (bottom) shows significant apoptosis in both WT and KO2 as well. (D) Serum alanine aminotransferase (ALT) levels are equivalent in WT and KO2 mice 6 h after GalN/LPS treatment (n ≥ 3). (E) IP shows maintenance of p65/β-catenin association in both WT and KO2 after GalN/LPS-induced injury.

Figure 3

Dissociation of the inhibitory p65/β-catenin complex after TNF-α coincides with initiation of DNA replication during acute liver injury and increased β-catenin activation in vitro. (A) IP shows that p65/β-catenin association is lost 3 h after TNF-α treatment in Hep3B cells but rebounds at 6 h. (B) p65/β-catenin association also decreases 3 h after GalN/LPS treatment. (C) Quantification of proliferating cell nuclear antigen (PCNA) staining shows a strong induction of cell cycle initiation 3 h after GalN/LPS treatment in WT livers, which is virtually absent at later time points. *p < 0.05. (D) Inhibition of p65 increases β-catenin/TCF4 reporter activity in Hep3B cells. **p < 0.01.

Figure 4

Despite increases in both p65 and β-catenin nuclear translocation, as well as increased cyclin D1 expression, the p65/β-catenin complex is unchanged during early liver regeneration. (A) IP shows insignificant changes in p65/β-catenin association after PH. (B) β-Catenin translocates to the nucleus 1 h after PH, while p65 increases in the nucleus 3 h after PH, as assessed by WB of nuclear lysates. (C) Real-time polymerase chain reaction (PCR) for cyclin D1 mRNA shows significant increases in cyclin D1 expression at both 1 and 12 h after PH. *p < 0.05; **p < 0.01. (D) WB shows a significant increase in cyclin D1 protein 12 h after PH.

Figure 5

Cyclin D1 activity and proliferation in vitro require β-catenin but not tumor necrosis factor-α (TNF-α)/p65. (A) p65 siRNA decreases p65 reporter activity, while β-catenin siRNA increases p65 activity. (B) β-Catenin siRNA significantly decreases cyclin D1 reporter activity in Hep3B cells, while p65 siRNA has no effect. (C) β-Catenin siRNA significantly decreases proliferation of Hep3B cells, as assessed by thymidine incorporation assay. (D) In actively proliferating cells expressing mutant S33Y β-catenin, siRNA against β-catenin, but not p65, decreases cyclin D1 reporter activity. (E) TNF-α induces p65 reporter activity in Hep3B cells. (F) Addition of TNF-α to Hep3B cells does not change cyclin D1 reporter activity. *p < 0.05; **p < 0.01.

Figure 6

β-Catenin/TCF4 regulates cyclin D1 expression in a p65-independent manner after PH, while in the absence of β-catenin, p65 target gene expression increases. (A) ChIP using TCF4 antibody shows an increase in cyclin D1 promoter occupancy 6 h after PH compared to baseline. (B) ChIP using p65 antibody demonstrates no difference in cyclin D1 promoter occupancy after PH. (C) cDNA analysis of NF-κB targets after PH shows that KO1 have a several-fold increase in several cytokine and apoptosis-related genes compared to WT.

Figure 7

NF-κB activation after partial hepatectomy (PH) occurs in nonparenchymal cells (NPCs) such as macrophages, while β-catenin activation occurs in hepatocytes. (A) Schematic of the NF-κB LacZ reporter mouse, which has three functional NF-κB sites upstream of a nuclear localization sequence and LacZ. (B) NF-κB activity is confined to NPCs before and immediately after PH, as assessed by β-galactosidase staining of histology sections. (C) β-Catenin IHC demonstrates activation of β-catenin (as determined by translocation into the cytoplasm and nucleus) predominantly in the hepatocytes at 3 and 6 h (3H and 6H, respectively) after PH. For (B) and (C), n ≥ 2 mice per time point.

Figure 8

Macrophages, which have a higher ratio of p65 to β-catenin than hepatocytes, produce Wnts after TNF-α stimulation. (A) WB of β-catenin and p65 shows that Hep3B cells have more β-catenin than p65, compared to Raw264 cells, which have more p65 than β-catenin. (B) IP for β-catenin shows presence of the p65/β-catenin complex in macrophages as well as Hep3Bs, despite low abundance of β-catenin. (C) RAW-Blue macrophages treated with TNF-α show a dose-dependent increase in NF-κB activity. **p < 0.01. (D) RAW-Blue cells show increased Wnt2 mRNA expression 6 and 24 h after incubation with a physiological dose of TNF-α. *p < 0.05.

Figure 9

Schematic for the role of β-catenin and NF-κB/p65 activation in liver injury and regeneration. (A) In acute liver injury, administration of LPS activates NF-κB in Kupffer cells, resulting in production of TNF-α, which in turn acts on neighboring hepatocytes to dissociate the p65/β-catenin complex and induce nuclear translocation of p65. (B) During liver regeneration, cytokines such as TNF-α activate NF-κB in Kupffer cells, resulting in expression of genes encoding growth factors as well as Wnt proteins; these factors are then released and act on neighboring hepatocytes in a paracrine manner to induce proliferation through transcription of cyclin D1.