Sustained phosphorylation of Bid is a marker for resistance to Fas-induced apoptosis during chronic liver diseases

Abstract

Background & aims: Increased rates of apoptosis have been reported to play a role in the pathophysiology of many disorders, including liver diseases. Conversely, genetic mutations that result in impairment of programmed cell death have been associated with cancer development. However, apoptosis resistance can also be the result of nongenetic stress adaptation, as seen in the cancer-prone metabolic liver disease hereditary tyrosinemia. To clarify whether stress-induced apoptosis resistance is a general feature of chronic liver diseases, an animal model of chronic cholestasis was examined.

Methods: Studies were performed with mice before and 2 weeks following bile duct ligation and with Fah-/- and Fah/p21-/- mice before and after NTBC withdrawal.

Results: Here we show that bile duct ligation induced profound resistance against Fas monoclonal antibody-mediated hepatocyte death. The apoptosis signaling pathway was blocked downstream of caspase-8 activation and proximal to mitochondrial cytochrome c release. In controls, activation of the Fas receptor resulted in rapid dephosphorylation of Bid and its subsequent cleavage, whereas Bid remained phosphorylated and uncleaved in chronic cholestasis and other models of hepatic apoptosis resistance.

Conclusions: We propose a model in which the phosphorylation status of Bid determines the apoptotic threshold of hepatocytes in vivo. Furthermore, resistance to apoptosis in chronic cholestasis may contribute to the long-term risk of cancer in this setting.

Figure 1

Bile duct–ligated mice develop a resistance against Fas-induced apoptosis. (A) Wild-type mice underwent BDL and were challenged with the Fas mAb Jo₂ (0.35 μg/g) 14 days later. Survival of control (squares) and bile duct–ligated (triangles) mice are shown. All control mice (n = 10) died from acute liver failure within 12 hours following injection of Jo₂, whereas all bile duct–ligated mice survived (n = 10). (B) DNA laddering following challenge with Jo₂ was only seen in control mice. Results are shown for duplicate animals under each condition. A standard DNA ladder marker (m) is shown. (C) TUNEL staining (brown color) of liver sections is shown (original magnification 200×) in control and bile duct–ligated mice before and after Jo₂ injection. TUNEL-positive cells were only visible in control mice following Jo₂ injection.

Figure 2

The mitochondrial pathway is blocked in bile duct–ligated mice. (A) Liver injury after Jo₂ challenge in control (WT; open columns) (n = 4) and bile duct–ligated mice (filled columns) (n = 4). Alanine aminotransferase and bilirubin levels were measured in plasma before (F.0) and after (F.4) challenge with Jo₂. Alanine aminotransferase levels increased only in control mice following Jo₂ injection. Bilirubin levels were significantly elevated in bile duct–ligated mice. Caspase-8 and caspase-3 activities were determined in liver homogenates 2 hours (F.2; caspase 8) or 4 hours (F.4; caspase 3) following Jo₂ injection (n = 4 each). In both groups, activation of caspase-8 was observed, whereas caspase-3 activity was only detectable in control mice. (B) Western blots of Bid, Bax, cytochrome c, and caspase-9 before (basal) and 4 hours after (Fas) induction of apoptosis with Jo₂ in control and bile duct–ligated mice (n = 4 each; 2 of 4 samples shown here) to determine activation of the mitochondrial pathway of apoptosis. Cleavage of Bid (decrease of full-length Bid), translocation of Bax, release of cytochrome c, and cleavage of caspase-9 following Jo₂ occurred only in control mice. F.0, before Fas mAb injection, F.2/F.4, 2/4 hours following Fas mAb injection.

Figure 3

Analysis of the mitochondrial pathway in wild-type and bile duct–ligated mice. (A) Western blot analysis of liver tissues in control and bile duct–ligated mice in the basal state without Fas mAb injection. Shown are data from 2 of 4 mice in each group. Liver tissues were analyzed for total cellular levels of Fas-R, Flip_s/l, cIap-2, Bax, Bid, Bak, Bcl-2, Bcl-x, and A1/Bfl-1. Of all the proteins analyzed, only A1/Bfl-1 was significantly induced 2 weeks following BDL. (B) Liver extracts of control and bile duct–ligated mice were incubated with in vitro activated recombinant caspase-8. Western blots for in vitro cleavage of caspase-3 and caspase-9 revealed no difference in both groups. (C) The mitochondrial fraction of control and bile duct–ligated mice was analyzed for BH-3–like proteins. Levels of Bak, Bcl-2, and Bcl-x were not significantly changed before and after challenge with Jo₂, whereas Bid was significantly increased in this fraction 2 weeks following BDL even before Jo₂ injection. (D) Alkaline extraction of mitochondria from bile duct–ligated mice and controls. Bid and Bax were only loosely attached and not inserted into the mitochondrial membrane of bile duct–ligated mice in contrast to Bcl-x. (E) Isolated liver mitochondria (n = 4 each; 2 of 4 samples shown here) were incubated with different concentrations of in vitro cleaved tBid, and translocation of cytochrome c from the mitochondrial (mito) fraction to the cytosol (released) was monitored by immunoblotting with a cytochrome c antibody. Cytochrome c release following incubation with 5 nmol/L tBid is shown. First row (−), spontaneous cytochrome c release without tBid incubation; second to ninth row, the overall mitochondrial cytochrome c content of the respective sample (n = 2 per group) is shown followed by the released cytosolic cytochrome c fraction following incubation with tBid. There was no difference in cytochrome c release between bile duct–ligated mice and controls.

Figure 4

Bid is not dephosphorylated following Fas injection in bile duct–ligated and in Fah^−/−/p21^−/− mice. (A) Phosphorylation of Bid at S61 was measured in cytosolic liver extracts from control and bile duct–ligated mice by Western blot. Increased phosphorylation of Bid in bile duct–ligated mice (n = 4 each; 2 of 4 samples shown here) before challenge with Jo₂ and, more importantly, no dephosphorylation following Jo₂ injection were seen. In contrast, Bid was almost completely dephosphorylated following induction of apoptosis in control mice. (B) Western blot analysis of CK1ε and PP2A levels in cell extracts from control and bile duct–ligated mice. The cytosolic CK1ε levels were higher in bile duct–ligated mice. Levels of PP2A were not changed in cytosolic extracts (data not shown). (C) CK1 activity was measured in cell extracts of control and bile duct–ligated mice with a CK1-specific peptide (n = 4). Note that activity assay is not specific for CK1ε but measures overall CK1 activity. CK1 activity was higher in bile duct–ligated mice at all time points. Interestingly, CK1 activity was reduced in control mice following challenge with Jo₂, but this reduction was not seen in bile duct–ligated mice. (D) Cellular extracts of control and bile duct–ligated mice following Jo₂ injection were analyzed for in vitro phosphorylation activity of His6-Bid (representative blot, n = 4 each); negative control (−) contains no cell extract, and positive control (CK1) contains recombinant CK1. Shown is a Western blot of the membranes probed with the phosphor-specific Bid (S61) antibody. Phosphorylation of Bid was only detectable in bile duct–ligated mice and was almost absent in controls. CK1 inhibitor (CKI-7) significantly inhibited phosphorylation of His6-Bid in bile duct–ligated mice. Total His6-Bid levels of non—CKI-7–treated reactions are shown. (E) Fah^−/− and Fah^−/−/p21^−/− mice were challenged with Jo₂ 2 weeks following NTBC withdrawal. Dephosphorylation and subsequent cleavage of Bid and cytochrome c release occurred only in the double-knockout mice. Total CK1ε levels were unchanged, whereas cytosolic levels were lower in Fah^−/−/p21^−/− mice following Jo₂ injection. (F) Wild-type mice were injected with Jo₂ alone or concomitantly with suramin. Suramin prevents dephosphorylation of Bid and subsequent Bid cleavage and cytochrome c release. Cytosolic CK1ε levels are higher in suramin-treated mice. (G) In suramin-treated mice, only a few scattered TUNEL-positive hepatocytes were visible following Jo₂ injection.

Figure 5

Atm phosphorylates Bid in vitro but is not necessary to prevent Fas-induced apoptosis during chronic liver diseases. (A) The ATM-p53 DNA damage pathway is activated in bile duct–ligated mice, measured by increased phosphorylation of p53 and increased overall levels of p53 and effector proteins p21 and Bax. (B) Recombinant Atm phosphorylated Bid-GST in vitro. (C) Bile duct–ligated Atm^−/− mice are protected against Fas mAb–induced apoptosis similar to wild-type mice. Dephosphorylation of Bid and subsequent cleavage of Bid, release of cytochrome c, and cleavage of caspase-9 and caspase-3 occurs only in wild-type mice. (D and E) TUNEL staining (brown color) of liver sections is shown (original magnification 200×). TUNEL-positive cells are only visible (D) in control mice 6 hours after Jo₂ injection but not (E) in Atm^−/− mice. (F) Cytosolic CK1ε levels are again higher in bile duct–ligated mice, whereas PP2A levels remain unchanged.