Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis

Abstract

The protein Bid is a participant in the pathway that leads to cell death (apoptosis), mediating the release of cytochrome c from mitochondria in response to signals from ′death′ receptors known as TNFR1/Fas on the cell surface^1–7. It is a member of the pro-apoptotic Bcl-2 family⁸ and is activated as a result of its cleavage by caspase 8, one of a family of proteolytic cell-death proteins. To investigate the role of Bid in vivo, we have generated mice deficient for Bid. We find that when these mice are injected with an antibody directed against Fas, they nearly all survive, whereas wild-type mice die from hepatocellular apoptosis and haemorrhagic necrosis. About half of the Bid-deficient animals had no apparent liver injury and showed no evidence of activation of the effector caspases 3 and 7, although the initiator caspase 8 had been activated. Other Bid-deficient mice survived with only moderate damage: all three caspases (8 and 37) were activated but their cell nuclei were intact and no mitochondrial cytochrome c was released. We also investigated the effects of Bid deficiency in cultured cells treated with anti-Fas antibody (hepatocytes and thymocytes) or with TNFα (fibroblasts). In these Bid^−/− cells, mitochondrial dysfunction was delayed, cytochrome c was not released, effector caspase activity was reduced and the cleavage of apoptosis substrates was altered. This loss-of-function model indicates that Bid is a critical substrate in vivo for signalling by death-receptor agonists, which mediates a mitochondrial amplification loop that is essential for the apoptosis of selected cells.

To assess the role of Bid in vivo, we constructed a targeting vector that deleted a 4.2-kilobase (kb) PstI/NheI Bid murine genomic fragment containing exons 1 and 2 and part of exon 3, which encodes the BH3 domain (Fig. 1a)⁹. Two of the independent, homologous recombinant clones of RW-4 embryonic stem (ES) cells transmitted the disrupted allele to the germline (Fig. 1b, c). The absence of Bid expression in Bid^−/− tissues was demonstrated by immunoblotting using a polyclonal anti-Bid antibody (Fig. 1d). Bid^−/− mice were born at the expected mendelian frequency, had no apparent developmental abnormalities, and had normal spleens, thymuses, brains, hearts, livers, lungs, kidneys and testes with respect to weight and histology (data not shown).

Figure 1.

Generation of Bid^−/− mice by gene targeting. a, Diagram of the Bid genomic locus, the targeting vector and the homologous recombinant. Also indicated are the restriction enzyme cutting sites (H; HindIII; P, Pst1; N, NheI; X, Xho1; B, BamHI) and the position of a 3′ external probe. Arrowheads labelled a, b and c indicate the positions of primers used in screening genotypes by PCR. b, Southern blot demonstrating homologous recombination within the Bid locus of the two ES clones that subsequently transmitted to the germ line. The 3′ external probe detects a 9.5-kb HindIII fragment for wild-type loci and a 8.2-kb HindIII recombinant fragment. c, Genotypes of mouse progeny using the same Southern strategy. d, Western blot demonstrating the absence of Bid protein but the presence of Bcl-2 and Bax in Bid^−/− mice.

The engagement of Fas/TNFR1 death receptors^10,11 leads to the formation of a protein complex known as DISC (or death-inducing signalling complex) and to the activation of caspase 8 (refs ^{12, 13}), which cleaves the larger (M_r 22K) cytosolic p22 Bid to its active form, p15 Bid, which translocates into the mitochondrion as an integral membrane protein^1–3. p15 Bid has been implicated as a component required for the release from mitochondria of cytochrome c (refs ^{1, 3}). To determine whether Bid is required in vivo for cytochrome c release, mitochondrial dysfunction or the death of cells, we injected mice intravenously with anti-Fas antibody (Jo2; 0.25 μg g⁻¹)¹⁴. Most wild-type (WT) mice (7/8) died within ~4h of acute liver failure associated with massive hepatic apoptosis and haemorrhagic necrosis (Fig. 2; Table 1). Within 2 h of injection, the initiator caspase 8 and the effector caspases 3 and 7 were activated, as evidenced by immunohistochemical staining of livers for caspases 3 and 7 (Figs 3b, 4b), immunoblotting (Fig. 3g) and cleavage of selective peptide substrates (Fig. 3h)¹⁵. Cytochrome c immunoreactivity was lost in wild-type hepatocytes undergoing apoptosis, reflecting either release and degradation or a conformational change in cytochrome c (Figs 3a, 4a)^16,17. In contrast, most Bid^−/− mice (9/11) survived anti-Fas antibody injection (Fig. 2a), about half had no apparent liver injury by gross or microscopic examination (Fig. 2b, c; Table 1), and there was no evidence of apoptosis from nuclear morphology (Fig. 3e) or TUNEL staining (Fig. 3f compared to 3c). Analysis of this subset of Bid-deficient livers indicated that caspase 8 had been activated (Fig. 3g, h) but that caspases 3 and 7 had not, as shown by analysis in situ (Fig. 3e), immunoblotting (Fig. 3g) and cleavage of the DEVD peptide substrate (Fig. 3h); nor had cytochrome c been released or its immunoreactivity lost in these Bid-deficient hepatocytes (Fig. 3d). These unaffected mice thus require a Bid-mediated pathway for effective effector-caspase activity and for mitochondrial dysfunction to cause apoptotic damage.

Figure 2.

Resistance of Bid^−/− mice to anti-Fas-antibody-induced mortality and hepatic injury. a, Survival curve of wild-type (WT; filled circles, n = 8) and Bid^−/− mice (circles, n = 11) in response to a single i.v. injection of 0.25 μg g⁻¹ anti-/Fas antibody (Jo-2). This represents the survival curve of the best-matched cohort from a total of 24 WT and 40 Bid^−/− mice that were injected with anti-Fas antibody. b, c, Haematoxylin–eosin staining of paraffin-embedded liver sections of WT (b) and Bid^−/− mouse (c) 2 h after anti-Fas antibody injection. Note the condensed and fragmented nuclei and the haemorrhage in the WT liver. Scale bar, 25 μm.

Table 1.

Resistance of Bid^−/− mice to anti-Fas-induced mortality and hepatocyte injury

Mice	Survival at 48 h (%)	Survival time of deceased animals (h) (mean ± s.d.)	2h (mean ± s.d.)	Histology scores* 2.5–3.5h	6–9 h
WT	12.5%	3.2 ± 1.3	1, 2, 3, 3†	3‡, 3‡	3‡
	(n = 8)	(n = 7	(2.3 ± 1.0)
Bid−/−	81.8%	6.6 ± 1.3	0, 0, 1, 0†	0†,1†	3‡
	(n = 11)	(n = 2)	(0.3 ± 0.5)
P ¶	<0.01	0.01	0.01	-	-

^*Semiquantitative histological scoring of liver injury (see Methods). Data shown are scores for each mouse and also mean ± s.d. for animals killed at the 2-h timepoint.

^†WT and Bid^−/− mice were killed at matched timepoints for comparison.

^‡Animals were killed when they became symptomatic.

^¶Statistics were performed using χ² test for survival rate, t-test for mean survival time and mean histology score.

Figure 3.

Lack of cytochrome c release, caspase activation or DNA fragmentation in unaffected Bid^−/− hepatocytes in response to anti-Fas antibody. a–f, Assessment of cytochrome c (a, d), activation of caspases 3 and 7, as detected by CM1 (ref. ³⁰) (b, e) and DNA fragmentation by TUNEL (c, f) in hepatocytes of WT (a–c) and unaffected Bid^−/− mice (d–f). Scale bar, 25 μm. Typically, onset of apoptosis in WT hepatocytes is accompanied by loss of cytochrome c staining (a). g, Analysis of the activation of caspases 8 and 3 in hepatocytes by immunoblot 2 h after injection of anti-Fas antibody. The p10 caspase-8 and p12 caspase-3 fragments represent the activated proteases. h, Caspase activity in S100 fractions prepared from hepatocytes 2 h after injection with anti-Fas antibody for WT (black bars) and unaffected Bid^−/− (white bars). Caspase activity was measured by release of the AFC fluorochrome from peptide substrates selective for caspase 8 (LETD), caspases 3 and 7 (DEVD), or the Bid site (LQTD). Data are mean :± s:d: of two mice from one of four representative experiments. The difference in the activity of caspases 3 and 7 between WT and Bid^−/− hepatocytes is statistically significant (P < 0:05).

Figure 4.

Altered pattern of apoptosis in affected Bid^−/− hepatocytes 2 h after injection of anti-Fas antibody. Comparison of a, e, cytochrome c status; b, f, activation of caspases 3 and 7 by staining with CM1 antibody³⁰; c, g, bisbenzimide nuclear morphology in the same liver section of Bid^+/+ (a–d) or affected Bid^−/− mice (e–h); d, h show three-colour immunohistochemical staining for cytochrome c (green), activated caspases 3 and 7 by CM1 (red) and nuclear morphology (blue). Scale bar, 25 μm.

Other Bid^−/− mice had moderate liver injury which they often survived; however, the two that died survived longer (6.6 h) than wild-type mice (3.2 h) (Fig. 2a; Table 1). Bid-deficient mice may suffer hepatocyte injury depending on the effectiveness of delivery of anti-Fas antibody or perhaps on the variable presence of a modifier locus. Using immunohistochemical analysis of affected regions of Bid^−/− livers, we found that, although caspases 3 and 7 were activated (Fig. 4f), cytochrome c immunoreactivity was often retained (Fig. 4e) and nuclei were not substantially condensed or fragmented (Fig. 4g) compared with wild-type livers (Fig 4a–c). Co-staining for activated caspases 3 and 7 and for cytochrome c, together with nuclear morphology, confirmed that damage in affected areas of Bid^−/− livers was mild. Wild-type hepatocytes had activated caspases 3 and 7 in the same cells that had lost cytochrome c immunoreactivity and their nuclei were condensed or fragmented (Fig. 4d); however, affected Bid^−/− hepatocytes activated caspases 3 and 7 in the presence of cytochrome c immunoreactivity and relatively normal nuclei (Fig. 4h). At this 2-h timepoint, the subcellular distribution of activated caspases 3 and 7 in Bid^−/− hepatocytes was largely restricted to the cell periphery (Fig. 4h).

We investigated the Bid-dependent component of apoptosis in cultured hepatocytes, embryonic fibroblasts (MEFs) and thymocytes. Cultured Bid-deficient hepatocytes survived for longer after treatment with anti-Fas antibody and cycloheximide, although they died later in the presence of cycloheximide (Chx) (Fig. 5a). Bid^−/− hepatocytes treated in vitro showed a delay in caspase-8 activation, corrected by 10 h, whereas the activity of caspase 3 was diminished throughout the time course of the experiment, as indicated by immunoblotting (Fig. 5b) and peptide cleavage (results not shown). Although MEFs and thymocytes do not absolutely require Bid for TNF-α/Fas-induced death, Bid deficiency delays apoptosis (Fig. 5c) and we were able to make a biochemical comparison. Bid-deficient thymocytes displayed more resistance to recombinant Fas ligand (FasL) as well as to anti-Fas antibody. Bid^−/− MEFs treated with TNF-α did not release substantial amounts of cytochrome c from mitochondria (Fig. 5d); caspase 8 was activated in Bid^−/− MEFs and thymocytes treated with TNF-α or anti-Fas antibody, respectively (Fig. 5e). Activation of caspases 3 and 7 was delayed and diminished in Bid-deficient MEFs and thymocytes, as assessed by immunoblotting (results not shown) and DEVDase activity (Fig. 5e). At later time points, Bid-deficient MEFs had less caspase-8-selective LET-Dase activity (Fig. 5e), which may reflect defective feed-forward cleavage of caspase-8 following onset of mitochondrial dysfunction¹⁸. There was less cleavage of some downstream substrates in Bid-deficient cells (Fig. 5f), including polyADP-ribose polymerase (PARP) and ICAD^19,20, which are nuclear components of the apoptosis pathway—this may bear on the retention of nuclear morphology in Bid^−/− cells that have started apoptosis (Fig. 4g, h). Cleavage of the cytosolic 14–3-3 protein was slightly delayed in Bid^−/− compared with +/+ cells, but was similar in both for the cytoskeletal fodrin molecule (Fig. 5f). These differences in the pattern and time course of substrate cleavage may relate to the peripheral distribution as well as the delayed activation of caspases 3 and 7 in Bid^−/− cells (Fig. 4f, h).

Figure 5.

Altered apoptotic program in anti-Fas-treated cultured hepatocytes and thymocytes or TNFα-treated embryonic fibroblasts (MEFs) from Bid^−/− mice. a, Viability of cultured hepatocytes from Bid^+/+ (filled triangles) or Bid^−/− (triangles) mice treated in vitro with anti-Fas antibody/cycloheximide. The spontaneous death rate of untreated hepatocytes is subtracted and the relative viability plotted. b, Activation of caspase 8 (p26 intermediate fragment) and caspase 3 (p17 fragment) at 4 and 10 h after treatment of hepatocytes with anti-Fas/cycloheximide (Chx). c, Cell viability of MEFs treated with TNFα/ActD versus staurosporine, and thymocytes treated with anti-Fas antibody or recombinant FasL. Loss of mitochondrial transmembrane potential, as assessed by the percentage of cells with low DiOC6 fluorescence, after treatment of WT (filled symbols) or Bid^−/− (triangles) MEFs with TNF/ActD or staurosporine and thymocytes with anti-Fas Ab/Chx or dexamethasone. Data shown for MEFs and thymocytes are from one of three representative experiments. d, Analysis of cytochrome c release in Bid^+/+ versus −/− MEFs after treatment with TNFα/ActD for 2, 4 or 8 h. The heavy membrane (HM) fraction is enriched for mitochondria (confirmed by anti-cytochrome oxidase antibody); the soluble S100 fraction (S) is the cytosolic component. Fractions were screened by western blot with anti-cytochrome c antibody. e, Time course of caspase activity in WT (black bars) or Bid^−/− (white bars) MEFs and thymocytes after treatment with TNFα/ActD or anti-Fas antibody/Chx, respectively. Data for MEFs and thymocytes are mean :± s:d: from duplicate samples from one of three representative experiments. Values for Bid^+/+ verus −/− MEFs at 8 h after treatment were significant for caspases 3 and 7 (P < 0:0005) and caspase 8 (P < 0:005), whereas thymocytes varied significantly for caspases 3 and 7 (P < 0:01), but not for caspase 8 (P > 0:05). f, Altered pattern of cleavage of caspase substrates within Bid^−/− versus Bid^+/+ thymocytes after treatment with anti-Fas antibody. Immunoblot detection of the cleavage of the cytoskeletal substrate fodrin was comparable in −/− and +/+ cells, whereas cleavage of ICADs and PARP, which participate in the nuclear programme, was delayed and diminished in Bid^−/− cells as was the 14–3-3 substrate.

Mitochondrial dysfunction occurs in the Bid-dependent pathway. In the absence of Bid to translocate to the mitochondria, the transmembrane potential (ΔΨ_M) of mitochondria is better maintained in Bid-deficient MEFs treated with TNF-α or in thymocytes treated with anti-Fas antibody (Fig. 5c). In contrast, other stimulators of cell death such as staurosporine, dexamethasone and γ-radiation (results not shown), which do not signal primarily through TNFR1/Fas, gave comparable alterations of ΔΨ_M and survival of Bid^+/+ and −/− cells (Fig. 5c).

Our Bid-deficient mice demonstrate that Bid is an indispensable substrate for select cells and death agonists, as shown by the hepatocyte apoptosis induced by Fas in vivo. The subset of null mice that had no liver injury was the most striking. Hepatocytes may resemble type-II cell lines in which Fas-induced death is dependent on mitochondria¹⁸. Consistent with this, a Bcl-2 transgene protected liver from Fas-induced apoptosis²¹, although this effect may be dose-dependent²². Cultured hepatocytes show more delay in activation of caspase-8 than do in situ hepatocytes, although both require a Bid-dependent mitochondrial loop to activate sufficient effector caspases.

Other Bid^−/− cells died, although they underwent a moderated pathway of apoptosis which defined a Bid-dependent component that affects mitochondrial function and cytochrome c release, the recruitment of effector caspases, selection of substrates and nuclear damage. The differential cleavage of substrates may reflect the amount and restricted distribution of effector caspases in the absence of a Bid-induced cytochrome c/Apaf-1/caspase-9 amplification loop²³, needed to generate a more effective and widely distributed activation of effector caspases. This may account in part for the decreased cellular injury and improved survival of Bid-deficient cells and mice. Retention of cytochrome c and the sustained transmembrane potential in null cells also implicate mitochondria in the Bid pathway. Death stimuli that do not use the TNFR1/Fas receptor are not much affected by Bid deficiency. Our Bid-deficient mice did not undergo any alteration in developmental cell death, as noted in knockouts of caspase 8 (ref. ²⁴), caspase 9 (refs ^{25, 26}) or Apaf-1 (refs ^{27, 28}). The Bid-dependent pathway also shows cell-type specificity, suggesting that pro-apoptotic Bcl-2 members²⁹ may act as selective links between specific upstream signals and downstream death effectors. Our results from null mice indicate that Bid may have evolved to trigger a mitochondrial amplification loop to ensure the death of cell types having limited access to initiator or effector caspases and/or in response to weak death-receptor signals. □

Methods

Generation of Bid^−/− mice.

A P1 murine genomic clone of 129/SvJ origin containing the Bid locus was identified. Restriction mapping and DNA-sequence analysis of subcloned fragments revealed that the murine Bid gene contains at least 5 exons and 4 introns spanning a region of at least 7.5 kb (ref. ⁹). A 5-kb 3′ fragment and a 2.1-kb 5′ fragment were cloned into a gene-replacement vector pNTK. The deleted 4.2-kb central region, replaced by a neomycin-resistance gene (neo), eliminates part of exon 1 containing the initiation methionine, exon 2, and part of exon 3, including the BH3 domain. The targeting vector was transfected into RW4 ES cells and neomycin-resistant clones were selected. Southern-blot analysis using a 3′ external probe recognizes a 9.5-kb HindIII wild-type fragment and an 8.2-kb HindIII homologous recombinant fragment. Three homologous recombinant RW4 clones were identified and two were injected into C57BL/6 blastocysts. Six chimaeric males from these two ES clones transmitted to the germ line. Mouse genotypes were determined by a three-primer PCR approach and confirmed by Southern blotting of genomic DNA isolated from tail biopsies. Western blots of lysates prepared from thymocytes and splenocytes were separated by 12.5% SDS–PAGE. Antibodies used were a polyclonal rabbit anti-murine Bid, a monoclonal anti-murine Bcl-2 (3F11), and a polyclonal anti-murine Bax (651) antibody. Secondary goat antibodies were conjugated with horseradish peroxidase (CALTAG). Blots were developed by ECL (Amersham).

Animal and cell-culture experiments.

Young adult mice were injected intravenously with anti-Fas antibody (clone Jo2; Pharmingen) at a dose of 0.25 μg g⁻¹. Animals were either observed over 48–72 h for mortality or killed at matched time points for liver histology or biochemical studies. Thymocytes were prepared from Bid^+/+ or Bid^−/− mice and cultured with control buffer, 1 μg ml⁻¹ anti-Fas antibody (clone Jo2), or 100 nM recombinant FasL plus 30 μg ml⁻¹ cycloheximide (Chx; Sigma) or 1 μM dexamethasone. Murine embryonic fibroblasts were prepared and treated with 1 ng ml⁻¹ TNF-α (Sigma) plus 2 μg ml⁻¹ actinomycin D (ActD; Sigma) or 4 μM staurosporine. Livers were perfused with calcium and magnesium-free Hank′s solution, followed by L15 medium containing 5 mM CaCl₂ and 0.5 mg ml⁻¹ collagenase H. Liver parenchymal cells were isolated from the dissected liver by low-speed centrifugation and cultured in William′s E medium containing 10% FCS. Following attachment for 2–3 h, hepatocytes were treated with anti-Fas antibody (0.5–1 μg ml⁻¹) plus Chx (10 μg ml⁻¹). Cells were analysed for viability, mitochondrial transmembrane potential (ΔΨ_M) and caspase activation.

Histology and immunohistochemical staining.

Livers were weighed, cut into small (<1.0 cm) pieces and fixed overnight at 4 °C in 4% paraformaldehyde in PBS, Bouin′s solution, or methanolic Carnoy′s fixative. The tissue was then dehydrated, paraffin-embedded and cut at 4-μm thickness. Haematoxylin-and-eosin stained sections were used for histopathological evaluation of hepatic injury. Slides were scored by a pathologist blinded to the genotype of the mice and the experimental conditions. Livers were graded on a scale of zero to three. Grade 0 corresponds to normal hepatic histology; 1, rare (<1 per 40× field) apoptotic nuclei; 2, moderate numbers of (>1 per 40× field) apoptotic nuclei, with no or only rare focal haemorrhages; 3, severe hepatic injury, with frequent apoptotic nuclei and marked haemorrhage. TUNEL staining was done on paraformaldehyde-fixed sections.

Immunohistochemical staining was done using tyramide signal amplification (NEN Life Science Products)³⁰. Cytochrome c immunoreactivity was detected with mouse anti-cytochrome c antibodies (Pharmingen), and activated caspase-3 and 7 immunoreactivity with an affinity-purified rabbit polyclonal antiserum (CM1). This antiserum immunohistochemically recognizes both activated caspases 3 and 7, but not their zymogenic forms³⁰. Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and direct TSA, using either fluorescein or cyanine-3-conjugated tyramine. For dual cytochrome c and CM1 immunolabelling, sequential TSA immunodetection was performed. Fluorescently labelled sections were incubated with bisbenzimide (Hoechst 33258) before coverslipping to stain cell nuclei. Fluoroscence was recorded with a Zeiss-Axioskop epifluorescence microscope and MC-100 camera system.

Caspase activation.

Caspase activity was assessed by the cleavage of site-selected tetrapeptide reporter substrates with the relative specificity of LETD-AFC (caspase 8), LQTD-AFC (caspase-8 cleavage site in Bid), DEVD-AFC (caspases 3 and 7) (Enzyme Systems Products). Lysates of hepatocytes or thymocytes containing 25–50 μg of protein were incubated at 37 °C with 50 μM peptide substrates for 30 min. Fluorescence generated by the release of the fluorogenic group AFC (aminotrifluoromethyl-coumarin) upon cleavage by caspases, was detected at the excitation wavelength of 400 nm and an emission wavelength of 505 nm with an FL500 microplate fluorescence reader (Bio-Tek). The signal strength representative of caspase activity was corrected for background and standardized to equivalent protein. Activated fragments of caspases 8 and 3 and other caspase substrates were detected by western blot. Liver, thymocyte and MEF lysates were prepared in 10 mM Tris-HCl, pH 7.2, 142.5 mM KCl, 5 mM MgCl₂, 1 mM EDTA and 0.25% NP-40 with protease inhibitors (0.2 mM PMSF, 0.1% aprotinin, 1 μg ml⁻¹ pepstatin, 1 μg ml⁻¹ leupeptin). Equal amounts of protein were size-fractionated by 8% or 15% SDS–PAGE, transferred to a PVDF membrane and immunoblots developed using polyclonal anti-Bid, anti-caspase-3, anti-caspase-8, anti-14–3-3 (Santa Cruz), anti-ICAD, anti-PARP (Chemicon) or anti-fodrin (Chemicon) antibodies. Secondary goat antibody was conjugated with HRP (CALTAG) and western blots were developed with ECL (Amersham).

Analysis of cell viability and mitochondrial transmembrane potentials.

Thymocytes or MEFs were assessed for viability after treatment by propidium iodide exclusion and/or staining with 0.5 μg ml⁻¹ annexin V in the presence of calcium for 30 min and analysis by flow cytometry. Viable cells were gated based on low annexin V staining and high forward light scatter. Cells were incubated with 40 nM of DiOC6 (ref. ²) (3,3′-dihexyloxacarbocynine iodide; Molecular Probes) for 30 min to determine mitochondrial transmembrane potential. Cells with DiOC6 staining as a reflection of decreased ΔΨ_M were detected by flow cytometry.