ARTICLE |
Correspondence to: Sabine Angermüller, Dept. of Anatomy and Cell Biology II, U. of Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany. E-mail: Sabine.Angermueller@urz.uni-heidelberg.de
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Summary |
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Tumor necrosis factor- (TNF
) induces apoptosis in hepatocytes only under transcriptional arrest induced by galactosamine (GalN). In this study we demonstrated the shuttle of the transcription factor NF
B (nuclear factor-kappa B) in the liver tissue of mice within 30 min4.5 hr hours after GalN/TNF
treatment. NF
B translocation from cytoplasm to the nucleus is initiated by its separation from the inhibitory I
B proteins which include I
B
, I
Bß, and I
B. Thirty minutes after GalN/TNF
administration, NF
Bp65 in hepatocellular nuclei becomes increasingly detectable and reaches its highest level after 2.5 hr. Then export back into cytoplasm begins but, surprisingly, approximately 30% of NF
B remains in the nuclear fraction and appears as an immunoprecipitate in the nuclei of apoptotic hepatocytes. Non-apoptotic hepatocytes do not show any reaction product in the nuclei 4.5 hr after treatment. Correspondingly, the amount of dissociated I
Bß decreases in the cytoplasm up to 2.5 hr and increases again afterwards, although it does not reach the level of the control samples. No evidence of I
Bß in the nuclei was found either immunocytochemically or biochemically. Caspase-3 activity, which is responsible for apoptosis, increases significantly after 3.5 hr. At that time, apoptotic hepatocytes can occasionally be observed and, 4.5 hr after GalN/TNF
treatment, constitute approximately 30% of the hepatocytes.
(J Histochem Cytochem 50:15991609, 2002)
Key Words:
NFB, I
Bß, caspase-3 activity, apoptosis, hepatocytes, GalN-sensitized TNF
-treated, mice
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Introduction |
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The transcription factor identified by B belongs to the multigene rel family that includes p65, c-rel, relB, p50, and p52 (
B is sequestered in cytoplasm by members of the inhibitory I
B protein family, which includes I
B
, I
Bß, and I
B (
and other cytokines, such as interleukin-1 (IL-1), lead to the activation of NF
B (
B proteins by the IKK (I
B kinase complex) (
B undergoes a second post-translational modification, ubiquitination, which targets the proteins for rapid degradation by the proteasome. The NF
B/I
B complex dissociates itself (
B enters the nucleus and activates a variety of genes that contain the
B consensus binding site. I
B
, but not I
Bß, is one of the target genes regulated by NF
B, (
B
enters the nucleus, associates with NF
B, and abolishes the NF
B/DNA interaction and, consequently, the transcriptional activities of NF
B (
B
is responsible for the transport of NF
B back to the cytoplasm using the leucine-rich nuclear export sequence (NES) that occurs in the carboxyl-terminal region of this protein (
Bß and I
B are involved in the sequestration of NF
B in the cytoplasm but, unlike I
B
, they do not participate in the nuclear export of NF
B (
After injection of TNF, apoptotic liver cell death is observed only under transcriptional blockade (
B would also activate the transcription of genes that encode cytoprotective proteins (
, neither specific DNA fragmentation (
The decrease in protein synthesis implies that apoptosis is caused by molecules that exist in an inactive form in the cell, and that death-triggering receptors such as TNF-R1 can activate this latent mechanism through a protein interaction cascade (20 kD), and one small subunit (
10 kD). Activation involves proteolytic cleavage into subunits that form heterotetramers with two large and two small subunits (
The purpose of this study was to investigate the shuttle of NFB up to the time when multiple apoptotic hepatocytes can be demonstrated after injection of TNF
into GalN-sensitized mice. The corresponding decrease and increase of I
Bß, which belongs to the inhibitor I
B proteins of NF
B, was examined. Furthermore, we investigated whether the first appearance of apoptotic hepatocytes corresponds to the significant increase of caspase-3 activity.
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Materials and Methods |
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Recombinant murine TNF was generously provided by Dr. G.R. Adolf (Boehringer Institute; Vienna, Austria).
Animals
This study was performed in compliance with the German Animal Protection Law under a permit issued by the state government (Karlsruhe, Germany). Male BALB/c mice (25 g) obtained from the animal facility of Charles River Laboratories (Sulzfeld, Germany) were kept on a standard laboratory diet with free access to water. Food was withdrawn 16 hr before the beginning of the experiments.
GalN (700 mg/kg) (Roth Chemicals; Karlsruhe, Germany) was given IP in a volume of 200 µl saline. Recombinant murine TNF (5 µg/kg) was injected into the tail vein in a volume of 300 µl saline containing 0.1% HSA 15 min after GalN administration. Injection of TNF
alone or GalN alone served as a negative control for the induction of apoptosis. The mice were anesthetized IP by xylazine hydrochloride 10 mg/kg (Rompun 2%; Bayer, Leverkusen, Germany) and ketamine hydrochloride 200 mg/kg (Ketamine 10%; WDT, Garbsen, Germany). Livers were harvested 0.5 hr, 1.5 hr, 2.5 hr, 3.5 hr, or 4.5 hr after treatment.
Fixation and Embedding
For immunohistochemical studies, the livers were fixed via the portal vein with a fixative containing 4% paraformaldehyde in 100 mM Pipes (piperazine-N,N'-bis [2-ethanesulfonic acid]) buffer at a pH value of 7.4 for 5 min. For light and electron microscopic studies, the livers were fixed with 0.25% glutaraldehyde in 100 mM Pipes buffer containing 2% sucrose at a pH value of 7.4 for 5 min. Fifty-µm sections of the fixed liver were postfixed with aqueous osmium tetroxide, dehydrated in graded ethanols, and embedded in Epon 812. LR White-embedded tissue did not obtain a postfixation.
Immunohistochemistry
Five-µm sections were obtained from paraffin-embedded blocks. Two commercial polyclonal antibodies were used: anti-NFBp65 (goat clone C-20; Santa Cruz Biotechnology; Santa Cruz, CA) and anti-I
Bß (rabbit clone Ab-1; Oncogene Research, Boston, MA). The best results were obtained with heat pretreatment in an electric pressure cooker set containing 10 mM sodium citrate buffer, pH 6, for 2 min. After the endogenous peroxidase was quenched with 3% aqueous H202, sections were transferred to a protein blocking serum for 1 hr, followed by incubation in a humid chamber at 4C overnight with (a) anti-NF
B p65 diluted 1:500 or (b) anti-I
Bß diluted 1:200. For visualization of NF
B, sections were incubated with a biotinylated secondary antibody donkey anti-goat IgG complex (Santa Cruz Biotechnology) diluted 1:20 for 30 min. For visualization of I
Bß, sections were incubated with HRP-conjugated goat anti-rabbit IgM complex (DAKO Diagnostika; Hamburg, Germany) for 90 min. Specific binding of NF
B was amplified using the ABC staining system of Santa Cruz. Sections were stained with peroxidase substrate for about 5 min without counterstaining. For negative controls, slides were incubated with TNB blocking buffer instead of the primary antibody.
Preparation of Cytoplasmic and Nuclear Extracts
For biochemical studies, livers were rinsed with saline via the portal vein, excised, weighed, and homogenized in a PotterElvehjem homogenizer (Braun; Melsungen, Germany) with a loose-fitting Teflon pestle at 1000 rpm with eight up-and-down strokes in ice-cold homogenization medium (250 mM sucrose, 5 mM MgCl2, 10 mM Tris-HCl, pH 7.4), called buffer A. After filtration, the homogenate was centrifuged at 600 x g for 10 min at 5C in a Beckman TJ-6 centrifuge (Beckman Instruments; Munich, Germany). To prepare the cytoplasmic extract the obtained supernatant was centrifuged at 100,000 x g at 4C for 30 min in a Beckman L5-65B ultracentrifuge (swinging-bucket rotor).
The nuclear extract was prepared according to a modified method of
Preparation of the Nuclei for Electron Microscopy
A part of the resuspended nuclei was immediately fixed with 1.5% glutaraldehyde in 100 mM Pipes buffer, pH 7.4, overnight. After centrifugation in an Eppendorf centrifuge for 3 min the pellet was embedded in 1.5% agar. This agar pellet was cut into small samples, postfixed with osmium tetroxide, dehydrated, and embedded in Epon 812. Ultra-thin sections were counterstained with lead citrate for 1 min and examined in a Philips EM 301 electron microscope.
Western Blotting Analysis
The protein quantity of cytoplasmic and nuclear extracts was performed using Bradford reagent (Biorad; Munich, Germany). Five µg protein for detection of NFBp65 and I
Bß separated in SDS-10% polyacrylamide gels were transferred to polyvinylidine difluoride membranes (Millipore; Eschborn, Germany) by electroblotting. Membranes were washed in PBS/Tween buffer (PBS containing 0.05% Tween-20) and incubated in blocking buffer containing a mixture of one part PBS/Tween-20 and one part non-fat milk (0.3% fat), followed by washing and incubation with the primary antibody which had been diluted (1:1000) in the same buffer overnight. Immunocomplexing was performed using the same primary antibodies as described for immunohistochemistry. The immunocomplexes were detected by using horseradish peroxidase-labeled donkey anti-goat antibody (Santa Cruz Biotechnology), or, respectively, goat anti-rabbit antibody, using the enhanced chemiluminescence (ECL) Western blotting detection system (NEN; Boston, MA). The intensity of immunoreactive bands was quantified by densitometry using NIH-Image software (version 1.59; National Institutes of Health, Bethesda, MD).
Measurement of Caspase-3 Activity
Caspase-3 activity was determined according to the method of
Light, Fluorescence, and Electron Microscopy
To investigate the typical morphological signs of apoptosis (the crescents of condensed heterochromatin along the nuclear envelope), we stained LR White-embedded semithin sections with hematoxylin for 10 min, followed by eosin for 1 min, or we stained with DAPI (4',6-diamine-2'-phenylindole dihydrochloride) in an aqueous dilution of 1:10,000 for 1530 min. DAPI-stained tissue was examined in the Zeiss/Axiophot and photographed on Kodak Elitechrome 400. For electron microscopic studies sections were treated as described above.
In Situ Cell Death Detection of DNA Strand Breaks by Fluorescence Microscopy
For in situ labeling of DNA strand breaks in nuclei we used LR White-embedded semithin sections of the livers of mice that had been treated with GalN/TNF for 4.5 hr, and applied the method of
Quantification of Apoptotic Cells
We counted the nuclei of hepatocytes in HE-stained semithin sections of the liver of mice (n=3) 4.5 hr after treatment with GalN/TNF. A total of 300 nuclei were randomly counted and the percentage of the nuclei that exhibited condensed crescent-shaped chromatin along the nuclear envelope was calculated.
Statistical Analysis
The results were analyzed by one-way ANOVA followed by the Dunnett Multiple Comparisons Test of the control against all other groups using the program InStat (Graph Pad Software; San Diego, CA). All data in this study were expressed as mean ± SEM. p0.05 was considered significant.
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Results |
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Purity of the Nuclear Fractions
The purified nuclear fractions of control livers exhibited good light and electron microscopic preservation without cytoplasmic contamination (Fig 1A and Fig 1B). Some nuclei of the fractions prepared 4.5 hr after GalN/TNF treatment revealed the typical apoptotic chromatin condensation (Fig 1C). Biochemically, we did not detect any NF
B in the control nuclear fractions (Fig 5). I
Bß was detected neither in the control nuclear fractions nor in the nuclear fractions resulting from GalN/TNF
treatment (data not shown). This may confirm the purity of the nuclear fractions. We have used this preparation procedure for the biochemical nuclear investigations.
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NFB
The shuttle of NFB was demonstrated by immunohistochemistry and Western blotting analysis, including quantitative analysis of the protein level based on the density of immunoreactive bands of Western blots of cytosolic and nuclear fractions.
Immunohistochemistry.
In the liver of control animals, the incubation with anti-NFB revealed immunoreactivity only in the cytoplasm of hepatocytes (Fig 2A). Replacement of the primary antibody by the blocking buffer TNB was used as a control for specific labeling. No reaction product was found in the cytoplasm of hepatocytes (Fig 2B). The translocation of NF
B into the nucleus began 30 min after GalN/TNF
administration (Fig 2C). Surprisingly, 4.5 hr after treatment immunoreactivity was observed in the heterochromatin of nuclei of apoptotic hepatocytes whereas, conversely, the reaction product could not be found in the nuclei of non-apoptotic hepatocytes. Diffuse staining of the cytoplasm showed the shuttle of NF
B back to the cytoplasm after 4.5 hr (Fig 2D).
Western Blotting Analysis and Quantitative Analysis of Labeling Density.
Western blotting analysis and quantitative analysis of the labeling density revealed a decrease of NFB in the cytoplasm, with the lowest level 2.5 hr after treatment. NF
B increased again but did not reach the level of the control fractions (Fig 4). Correspondingly, the NF
B content in the nuclear fractions reached its maximum 2.5 hr after GalN/TNF
treatment and decreased to approximately 30% 4.5 hr after treatment. In the nuclear fractions of control livers, NF
B could not be determined (Fig 5).
IBß
NFB is sequestered in the cytoplasm by the inhibitory I
B protein family, which includes I
B
, I
Bß, and I
B. We investigated I
Bß, which did not participate in the shuttle of NF
B.
Immunohistochemistry.
We documented the expression of IBß in the liver of control animals as a strong immunoreactivity in the cytoplasm of hepatocytes (Fig 3A). Replacement of the primary antibody by blocking buffer TNB was used as a control for the specific labeling. No reaction product was found in the cytoplasm of hepatocytes of control liver (Fig 3B). Ten min after stimulation the reaction product was still observed in the cytoplasm (Fig 3C) but could no longer be detected within 30 min2.5 hr after GalN/TNF
treatment (data not shown). However, 3.5 hr after stimulation I
Bß was found again in the cytoplasm (Fig 3D).
Western Blotting Analysis and Quantitative Analysis of Labeling Density.
At 42.5 hr after treatment, Western blotting analysis and quantitative analysis of the labeling density showed a decrease of the cytosolic IBß protein level to approximately 50% in comparison with the protein quantity of the control fractions. After this time the protein level increased again but did not reach the quantity of the control fractions (Fig 6). I
Bß was detectable neither in the nuclear fractions nor in the nuclei of tissue sections, indicating that I
Bß was not involved in the NF
B shuttle.
Caspase-3 Activity
Reduced synthesis of protective proteins by injection of GalN induced apoptotic cell death in hepatocytes of mice treated with TNF. Key mediators that initiated the apoptotic program were members of the caspase family. AMC fluorescence, the indication of caspase-3 activity measured by spectrofluorophotometry, increased perceptibly 2.5 hr after treatment and was more than 5 times higher 3.5 hr after treatment compared to the fluorescence measured in control fractions. 4.5 hr after stimulation, fluorescence increased to such an extent that it could no longer be measured. Peptid aldehyde, like DEVD-CHO, binding strongly to the caspase-3 active site and blocking the cleavage of the AMC group was used as specific control. This inhibitor reduced the activity, as measured 3.5 hr after treatment, to the level demonstrated in the control fractions (Fig 7).
Histological and Electron Microscopic Findings
We used LR White-embedded liver tissue for the morphological studies of apoptosis. In contrast to epon-embedded material, LR White-embedded semithin sections of liver tissue were regarded as appropriate for hematoxylin/eosin and for DAPI staining. Apoptotic cell death is very rare in healthy adult mouse liver (Fig 9A). Approximately one to five apoptotic cells/10,000 hepatocytes are found in untreated mice (for review see , it was hardly toxic in the applied dose (
induced hepatocellular apoptosis 4.5 hr after administration (Fig 9B and Fig 9C). Administration of GalN produced uridine diphosphate hexosamine complexes (
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Activated executioner caspase-3 led to apoptotic cell death. In accordance with the enormous increase of caspase-3 activity 3.5 hr after treatment, apoptotic hepatocytes appeared occasionally. These apoptotic cells were still surrounded by morphologically intact liver tissue. The typical shrinkage of the apoptotic cell, together with the well-known heterochromatin condensation along the nuclear envelope, is shown in Fig 8. The electron micrograph revealed mitochondria in which the outer membrane was scarified and the inner membrane had expanded through this gap, forming a herniated structure. Approximately 30% of the hepatocytes quantified in semithin liver sections 4.5 hr after treatment exhibited apoptotic characteristics (Fig 9D).
In Situ Cell Death Detection
The widely used TUNEL method end-labeled double-stranded, low molecular weight DNA fragments and single-strand breaks in high molecular weight DNA. The polymerization of dUTP to the 3'-hydroxyl end of fragmented DNA resulted in a fluorescent reaction product, which was detected in the apoptotic nuclei of hepatocytes of LR White-embedded semithin sections (Fig 10C). For controls, the negative control of an untreated animal (control 1) and the specificity control of a treated animal (control 2) did not reveal any reaction product (Fig 10A and Fig 10B). Apart from the TUNEL reaction, DAPI staining of tissue sections, embedded in LR White was also very useful for detection of apoptotic hepatocytes (Fig 10D).
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Discussion |
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In a previous study we showed evidence of apoptosis in hepatocytes 4.5 hr after injection of TNF into GalN-sensitized mice (
activate NF
B by releasing it from the cytoplasmic inhibitor protein I
B. Activated NF
B translocates into the nucleus, where it plays an important role in the regulation of genes that play a role in the immune response and in inflammatory processes (for review
B also activates cytoprotective genes of the IAP (inhibitor of apoptosis) family, which act as anti-apoptotic factors and support cell survival (
B between cytoplasm and nucleus and the decrease and increase of its inhibitor I
Bß in the cytoplasm of hepatocytes within 30 min4.5 hr after injection of TNF
into GalN-sensitized mice. In addition, we have shown that the first appearance of apoptotic hepatocytes is accompanied by a significant increase in caspase-3 activity.
NFB is sequestered in cytoplasm by the inhibitor protein I
B family, which includes I
B
, I
Bß, and I
B. I
Bß has a high affinity towards NF
B dimers by binding to the nuclear localization signals (NLS) of both NF
B subunits, whereas I
B
contacts only the NLS of the transcriptionally active p65 subunit of the p50p65(relA) heterodimer (
B appears to sequester p65 and/or c-rel in cytoplasm (
B
is rapidly degraded, whereas I
Bß and I
B are degraded with slower kinetics (
Bß in the cytoplasm is less (45%) than the reduction of NF
B in cytoplasm (65%). This suggests that NF
B bound to the other I
B molecules, such as I
B
and I
B, was also released from these inhibitors. Activated NF
B leads to the transcription of the I
B
gene, followed by a rapid re-synthesis of this protein. Newly synthesized I
B
enters the nucleus, combines with NF
B, and transports the transcription factor back to the cytoplasm (
Bß mRNA is not upregulated by NF
B (
Bß and I
B do not participate in the shuttle of NF
B (
Bß, confirming that I
Bß is purely cytosolic.
B into nuclei of hepatocytes 1 hr after treatment with GalN/TNF
, and a decrease of NF
B in the nuclei 5 hr after treatment. We determined NF
B in nuclei just 30 min after treatment. The maximal protein level in the nuclear fractions is reached 2.5 hr after GalN/TNF
administration, followed by an incomplete shuttle back to the cytoplasm after 4.5 hr. Approximately 30% of NF
B remained in the nuclear fractions. Immunohistochemical investigation revealed that NF
B was left in the nuclei of apoptotic hepatocytes. The quantification of apoptotic hepatocytes showed that about 30% of the parenchymal cells were apoptotic. It is likely that the inhibition of the protein synthesis after GalN injection also concerns the re-synthesis of I
B
. Therefore, in some hepatocytes a part of NF
B could not be transported back into the cytoplasm by I
B
. Whereas these hepatocytes were predestined for apoptotic cell death, the transport of NF
B back to cytoplasm rescued hepatocytes that survived. It should be noted that NF
B could not be found in the nuclei of non-apoptotic hepatocytes 4.5 hr after GalN/TNF
treatment. GalN depleted hepatic uridine nucleotides and formed uridine diphosphate hexosamine complexes (
Apoptosis is caused by activation of the initiator caspases-8 and caspase-9, which trigger executioner caspase-3 (B activity, apoptosis takes place (
B, appeared to be disturbed because the caspase cascade was initiated 2.5 hr after GalN/TNF
administration, as documented by the increasing AMC fluorescence that indicates caspase-3 activity. At this time apoptotic hepatocytes could not yet be found in liver sections. Similar to the study of
treatment and apoptotic hepatocytes could occasionally be seen. Caspase-3 activity furthermore increased enormously, so that approximately 30% of the hepatocytes were apoptotic 4.5 hr after stimulation. This remarkable increase in caspase-3 activity was probably caused by an amplification loop in which caspase-3 cleaves and activates pro-caspase-9 molecules (
In summary, we demonstrated that the shuttle of NFB back to the cytoplasm is disturbed after GalN/TNF
treatment. Approximately 30% of NF
B remained in the nuclear fractions and could be observed in the heterochromatin of the nuclei of apoptotic hepatocytes. We suggest that the re-synthesis of I
B
was blocked in hepatocytes in which large amounts of uridine diphosphate hexosamine complexes are contained. We found that the appearance of apoptotic hepatocytes was correlated to the enormous increase of caspase-3 activity. The interaction between c-IAP2 and caspase-3 may be disturbed and free caspase-3 can then display its activity.
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Acknowledgments |
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Supported by the grant An 192/2-1 by the Deutsche Forschungsgemeinschaft, Bonn Bad-Godesberg.
We thank Prof Dr A. Völkl, Prof Dr W. Plaga, and Dr Renate Bang for helpful discussions. The excellent technical assistance of Ms Annette Stradtmann is gratefully acknowledged.
Received for publication December 17, 2001; accepted June 26, 2002.
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