The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis

Abstract

The accumulation of bile acids during obstructive cholestasis causes liver injury and fibrosis, which is at least partly mediated by the death receptors Tumor necrosis factor-related apoptosis-inducing ligand, Tumor necrosis factor-alpha, and Fas. The BH3-interacting domain death agonist Bid is a critical mediator of death receptor-induced apoptosis in hepatocytes. Our aim for this study was, therefore, to elucidate whether Bid also mediates death receptor-induced liver injury in obstructive cholestasis. Overall, survival and various aspects of liver injury were analyzed in wild-type and Bid(-/-) mice after bile duct ligation (BDL), a commonly used model to study obstructive cholestasis in mice. Liver injury was examined at 3, 7, and 14 days after BDL. Loss of Bid did not affect the number of bile infarcts, serum aspartate aminotransferase values, or animal survival. Processing of procaspase-3 and procaspase-9, and caspase-3 enzyme activities, were not detectable in either group, and Bid(-/-) mice displayed the same pattern of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive hepatocytes as wild-type controls following BDL. In contrast to Fas-receptor deficient lpr mice, hepatic fibrosis and the inflammatory response was not affected by loss of Bid. Together, these data suggest that Bid is not a downstream target of death receptors in obstructive cholestasis and does not significantly contribute to bile acid induced liver injury and fibrosis.

Figure 1

A: Wild-type (WT) and Bid^−/− mice underwent BDL and were euthanized after 3, 7, and 14 days. Serum levels of AST and bilirubin as markers of hepatic damage were measured at indicated time points. Mean values and SD are presented for all time points (n = five in each group). B: H&E staining revealed profuse liver damage and multiple oncotic bile infarcts in BDL mice. C: Survival of WT and Bid^−/− mice was recorded after BDL (n = 20 mice in each group).

Figure 2

A: TUNEL and cleaved caspase-3 staining of liver sections is shown from wild-type (WT) and Bid^−/− mice after BDL and in mice injected with the Fas mAb Jo-2 (original magnification, ×200, n = four in each group). B: Hepatic caspase-3 activity was measured by using the CaspACE Assay kit and processing of caspase-9 (loss of full length caspase-9) and caspase-3 (detection of the cleaved fragment) was analyzed by Western blot for all time points before and after BDL and in Jo-2 injected mice as a positive control (+). C: Liver tissues of control and BDL mice were analyzed for total cellular levels of Fas, Flip, and Bid (n = 5, pooled samples).

Figure 3

A, B: Hepatocyte proliferation was assessed with BrdU immunostaining and BrdU positive cells were quantified (10 microscope fields at ×200 magnification, wild-type (WT) mice are represented as open bars and Bid^−/− mice as closed bars, n = 5). C: Immunoblots were performed for cyclin D and E and p21 (n = 5, pooled samples).

Figure 4

A: Liver fibrosis was evaluated by α-SMA, Sirius red, and Masson’s trichrome staining 3, 7, and 14 days after BDL (Original magnification, ×200). B: Hydroxyproline content measured in the right median (#3) and the right lateral (#5) liver lobes 7 days after BDL. C: mRNA levels of transforming growth factor-β, α-SMA, and matrix metalloproteinase-3 were quantified by real-time RT PCR (sham operated mice are represented with open bars and BDL mice with closed bars).

Figure 5

A: Liver sections of control and BDL mice were analyzed for CD68 expression and CD11b expression (Original magnification, ×200). Infiltrating neutrophils were also detected by (arrows) Naphthol ASD staining. B: Quantitative RT-PCR was performed by using pooled RNA extracted from wild-type (WT) and Bid^−/− mice before (open bars) and after BDL (closed bars) (n = 4). Specific cDNAs of KC, monocyte chemotactic protein-1, interleukin-6, and TNF-α were amplified.

Figure 6

A: Wild-type (WT) and Lpr mice underwent BDL and were euthanized after 3 days (n = 4). Liver injury was analyzed by H&E staining. B: Western blot of p-Erk, p-Jnk, p-p38, and c-jun in WT and Bid^−/− mice before and 3, 7, and 14 days after BDL. C: Analysis of p-Erk, p-Jnk, p-p38, and c-jun in WT and Lpr mice before and 3 days after BDL (positive control (+) for p-Jnk: WT mouse injected with Jo-2; positive control (+) for p-p38: Fah^−/− mouse injected with homogentisic acid). D: Representative H&E staining of control and c-jun^−/− mice 3 days after BDL. E: Serum AST and bilirubin levels were determined before and 3 days after BDL.