Autocrine expression of activated transforming growth factor-beta 1 induces apoptosis in normal rat liver

Laura W. Schrum1, Mark A. Bird1, Olga Salcher2, Elmar-Reinhold Burchardt2, Joe W. Grisham3, David A. Brenner4,5, Richard A. Rippe4, and Kevin E. Behrns1

Departments of 1 Surgery, 4 Medicine, 5 Biochemistry and Biophysics, and 3 Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7210; and 2 Bayer Pharmaceuticals, Wupertal 20000, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The aim of this study was to determine the differential effects of latent and activated transforming growth factor (TGF)-beta 1 in growth control of normal and proliferating hepatocytes in vivo. Rats were injected with adenoviruses expressing control transgenes (Ctrl), latent TGF-beta 1 [TGF-beta (L)], or activated TGF-beta 1 [TGF-beta (A)]. Additional animals underwent two-thirds partial hepatectomy (PH) 24 h after injection. Increased hepatocyte apoptosis was observed in TGF-beta (A)-injected but not TGF-beta (L)-injected animals 24 h postinjection (10.5%) compared with Ctrl animals (0.37%). The percent of apoptotic cells increased to 32.1% in TGF-beta (A)-injected animals 48 h after injection. Furthermore, TGF-beta (A)-injected rats did not survive 24 h after PH. Four hours after PH, 0.25 and 14.1% apoptotic hepatocytes were seen in Ctrl- and TGF-beta (A)-injected rats, respectively. TGF-beta (A)-induced apoptosis in primary rat hepatocytes was blocked with a pancaspase inhibitor. Thus autocrine expression of TGF-beta (A) but not TGF-beta (L) induces hepatocyte apoptosis in the normal rat liver. Rats overexpressing TGF-beta (A) do not survive two-thirds PH due to hepatic apoptosis. Thus activation of TGF-beta 1 may be a critical step in the growth control of normal and proliferating rat hepatocytes.

hepatic regeneration; caspase activity; growth factors; adenovirus; partial hepatectomy


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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IN THE HEALTHY LIVER hepatocytes divide rarely, but after chemical or physical injury hepatocytes progress from the G0 phase to the G1 phase of the cell cycle. Growth factors regulate this process through both stimulatory and inhibitory signaling. Epidermal growth factor (31), transforming growth factor (TGF)-alpha (32), and hepatocyte growth factor (34, 53) stimulate hepatocyte DNA synthesis, whereas TGF-beta 1 and interleukin-1beta inhibit hepatocyte replication (16). TGF-beta controls hepatocyte growth after partial hepatectomy (PH) through an autocrine feedback mechanism (5). TGF-beta has been investigated as a growth inhibitor of both hepatocytes in culture and in the regenerating liver, but the mechanism of growth control and the importance of relative abundance of activated [TGF-beta (A)] vs. latent [TGF-beta (L)] TGF-beta in normal and proliferating hepatocytes has not been examined thoroughly.

TGF-beta (A) inhibits hepatocyte DNA synthesis both in culture (35, 48) and in vivo (40). In primary hepatocyte cultures, TGF-beta 1 inhibits DNA synthesis from normal and regenerating livers by blocking the transition from the G1 to the S phase of the cell cycle (35, 48). After a two-thirds PH, TGF-beta 1 mRNA expression increases (27), and TGF-beta is the ostensible inhibitory peptide for hepatocyte replication and liver regeneration. Other in vivo studies, however, have demonstrated that mature TGF-beta administered intravenously reduced [3H]thymidine incorporation in hepatocytes after a PH, but inhibition of hepatocyte DNA synthesis was transient because complete regeneration of the liver still occurred by 8 days (40). In a transgenic mouse model overexpressing bioactive TGF-beta 1, administration of the latency-associated peptide (LAP) region of TGF-beta 1 prevented inhibition of hepatocyte DNA synthesis (6).

Both cell cycle arrest and apoptosis have been implicated as mechanisms of TGF-beta -induced hepatocyte growth arrest. TGF-beta has been shown to inhibit progression of the cell cycle by decreasing the expression of G1 cyclins and cyclin-dependent kinases (18). In rat liver epithelial cell lines, TGF-beta impairs DNA synthesis by preventing hyperphosphorylation of the retinoblastoma gene product (pRb; see Ref. 50). Moreover, in vivo administration of TGF-beta in the regenerating rat liver decreased pRb expression, suggesting that this growth-inhibiting cytokine played a prominent role in growth control of proliferating hepatocytes (15). Inhibition of hepatocyte proliferation, however, has also been attributed to apoptosis (37, 38). Only the bioactive form of TGF-beta 1, and not the latent form, induces apoptosis (37). However, this induction of apoptosis by TGF-beta (A) in vivo was shown only in liver undergoing cyproterone-induced hyperplasia (36) or in diseased liver (42). Therefore, the in vivo mechanism of hepatocyte growth control in the normal and regenerating liver remains to be determined.

TGF-beta 1 is translated as a latent dimeric precursor, and upon cleavage and dissociation of the amino terminal portion, known as LAP, a mature, biologically active protein is formed (19). TGF-beta 1 is secreted by hepatocytes in the latent form and is activated extracellularly by a poorly understood mechanism(s). Potential in vivo mechanisms of TGF-beta activation include plasmin (29), thrombospondin (TSP)-1 (44, 45), binding to the mannose 6-phosphate/insulin-like growth factor type II receptor (14), and the integrin alpha vbeta 6 (33), but these activation pathways may be tissue specific. Expression of TGF-beta 1 is regulated presumably through an autocrine feedback mechanism (5). TGF-beta signals through two active receptor subtypes, TGF-beta R1 and TGF-beta R2. TGF-beta binds to TGF-beta R2, which phosphorylates and activates TGF-beta R1. TGF-beta R1 acts as a serine-threonine kinase that activates the intracellular Smad family of signal transducers (23, 51).

Our study investigated the differential effect of autocrine expression of TGF-beta (L) and TGF-beta (A), delivered by adenoviral vectors, on hepatocyte growth control in the normal and proliferating hepatocytes in vivo. In this study, TGF-beta (A) induced hepatocyte apoptosis in the normal and regenerating liver, and overexpression of bioactive TGF-beta 1 was lethal to rats subjected to PH. Additional experiments showed that TGF-beta (A)-induced apoptosis in primary rat hepatocytes can be inhibited with a pancaspase inhibitor, suggesting that hepatocyte apoptosis and not cell cycle arrest is the major mechanism of TGF-beta 1-induced hepatocyte growth control. TGF-beta (L), however, did not alter hepatocyte DNA synthesis or induce apoptosis. These findings suggest that activation of TGF-beta 1 is a critical step in rat hepatocyte growth regulation after PH.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Adult male Sprague-Dawley rats (200 g) were fed a standard chow diet, had access to water ad libitum, and were kept under 12:12-h light/dark cycles. All animal procedures and protocols were in accordance with the National Institutes of Health guidelines and were approved by The University of North Carolina's Institutional Animal Care and Use Committee.

Purification of adenovirus. The recombinant replication-deficient adenoviruses [adenoviruses expressing beta -galactoside or luciferase (Ctrl), TGF-beta (L), or TGF-beta (A)] were grown in 293 cells and were purified by CsCl gradients (52). TGF-beta (L) adenovirus contains a 2.1-kb human TGF-beta 1 transgene and is converted to the bioactive TGF-beta 1 as demonstrated by the mink lung cell growth inhibition assay (49). TGF-beta (A) contained the full-length porcine TGF-beta cDNA with cysteine-to-serine mutations at positions 223 and 225, resulting in expression of biologically active TGF-beta 1 (46). Viral titers were determined by optical density and plaque assay (30). The recombinant virus was stored in 25% (vol/vol) glycerol at -80°C.

Adenoviral infection and PH. Adenoviruses were dialyzed against three changes of 1× PBS containing 1 mM MgCl2 at 4°C. The adenoviral vectors were injected via the tail vein [1 × 1010 plaque-forming units (PFU)] or intraportally (6.0 × 109 PFU) into 200-g adult male Sprague-Dawley rats. Twenty-four hours after adenoviral injection, a two-thirds PH was performed (25) under ketamine/acepromazine anesthesia. The remnant livers were harvested 4 or 24 h after PH. Livers were snap-frozen in liquid nitrogen or fixed in 10% neutral buffered formalin for histological analysis. Additional rats received TGF-beta (A) but did not undergo PH, and livers were harvested 6, 12, 18, 24, and 48 h after injection.

Infection of primary rat hepatocytes. Primary rat hepatocytes were isolated from adult male Sprague-Dawley rats by collagenase perfusion (22). Hepatocytes were plated in 60-mm tissue culture dishes (1.5 × 106 cells/plate) or 100-mm dishes (4 × 106 cells/plate) and were cultured in Waymouth's media containing 10% FCS, 5 µg/ml insulin, and 10-7 M dexamethasone in a 5% CO2-95% air atmosphere for 4 h. Cells were serum starved in hormonally defined medium (HDM; RPMI 1640 containing 5 µg/ml insulin, 10 µg/ml transferrin, 3 × 10-8 M selenium, and 10 nM free fatty acids) for 2 h. Cells were infected with either Ctrl or TGF-beta (A) adenoviruses at a multiplicity of infection of 30 for 2 h. Media was changed to fresh HDM with or without 5 µM Z-VAD.FMK (Calbiochem, La Jolla, CA). Cells were harvested at 18, 24, and 36 h for total RNA to determine TGF-beta (A) expression, fluorescence-activated cell sorter (FACS) and DNA isolation for apoptotic analysis, and whole cell extracts for caspase activity.

RNA isolation, Northern blot analysis, and beta -galactosidase and luciferase assays. Total RNA from the snap-frozen liver was isolated by CsCl ultracentrifugation (41). RNA samples (10 µg) were run on a formaldehyde gel (41) and transferred to a magna charge nitrocellulose membrane (MSI, Westborough, MA) by capillary transfer overnight. RNA was fixed to the membranes by ultraviolet cross-linking using a Stratalinker (Stratagene, La Jolla, CA) and was prehybridized [5× saline-sodium citrate (SSC), 5× Denhardt's, 50 mM sodium phosphate, pH 6.5, 0.1% SDS, 250 µg/ml fish sperm DNA, and 50% formamide] for at least 2 h at 42°C. Human TGF-beta 1 cDNA was labeled using the Rediprime Kit (Amersham, Arlington Heights, IL) as recommended by the manufacturer, 106 counts · min-1 · ml-1 was added to the prehybridization buffer, and incubation was continued for 15 h at 42°C. Blots were washed with 2× SSC and 0.1% SDS two times at room temperature for 5 min and then with 0.1× SSC and 0.1% SDS at 65°C for 20 min. Membranes were exposed to Biomax-MR film (Eastman Kodak, Rochester, NY) overnight at -80°C with an intensifying screen. Additionally, total RNA was isolated from hepatocytes by the phenol-guanidinium method (39) and was analyzed for TGF-beta (A) expression as described above.

Livers from Ctrl animals were analyzed for beta -galactosidase or luciferase activity. beta -Galactosidase assays were performed using the substrate 2-nitrophenyl-beta -D-galactopyranoside (41). To determine luciferase activity, livers were homogenized in luciferase cell lysis buffer (Analytical Luminescence Laboratory, San Diego, CA), and samples were rotated for 30 min at 4°C and centrifuged at 16,000 g for 10 min at 4°C. Luciferase assay was performed as described previously (43).

RNase protection assay. Total RNA was harvested from liver. Radiolabeled riboprobes for the RNase protection assay were derived from the 375-nt Pst I-Ava I fragment of the rat alpha 1(I) collagen cDNA. The riboprobe for the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was generated from the plasmid pTRI-GAPDH-Rat (Ambion, Austin, TX), which was linearized using Hind III. The radiolabeled probes were mixed with 50 µg of total liver RNA, and the samples were dried. Dried pellets were suspended in 30 µl hybridization buffer (100 mM PIPES, pH 6.7, 400 mM NaCl, 2 mM EDTA, and 80% formamide), heated at 85°C for 10 min, and incubated overnight at 45°C. The hybridization reaction was then incubated with 350 µl RNase buffer (300 mM NaCl, 10 mM Tris · HCl, pH 7.6, 40 mg/ml RNase A, and 2 mg/ml RNase T1) at 30°C for 1 h. Afterwards, 10 µl of 20% SDS and 5 µl of 10 mg/ml proteinase K were added, and the reaction mixture was incubated at 37°C for 15 min. The reaction mixture was phenol extracted and precipitated with the addition of 1 µg of yeast tRNA and 1.0 ml of 100% ethanol. The samples were suspended in formamide dye (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol FF) and heated at 95°C for 5 min and were loaded onto a standard 5% sequencing gel. After electrophoresis, bands were visualized by autoradiography and quantitated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).

Histology and immunohistochemistry. Paraffin-embedded tissues were sectioned at 5 µm thickness and stained with hematoxylin and eosin (H&E) for identification of necrosis and inflammation. For immunohistochemistry, paraffin sections were deparaffinized by three 5-min incubations in xylene followed by 5-min incubations in 100, 95, and 70% ethanol and rehydrated in water and PBS. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed using the in situ cell death detection kit (Boehringer Mannheim, Indianapolis, IN). Briefly, DNA ends were tagged with fluorescein-labeled dUTP using terminal deoxynucleotidyl transferase by incubating the samples at 37°C in a humidified chamber. Liver sections were then incubated with anti-fluorescein-alkaline phosohatase (AP) conjugate for 30 min in a humidified chamber. Slides were incubated with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma, St. Louis, MO) for 20 min at room temperature and were counterstained with hematoxylin. AP-positive cells were counted in five high-power fields (×400) per section, and the number of stained hepatocytes was expressed as a percentage of the total number of hepatocytes.

Caspase assay. Caspase 3-like and caspase 8-like activities were determined by measuring the in vitro fluorogenic peptide substrate carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin and carbobenzoxy-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethyl coumarin, respectively, as described by the manufacturer (Bio-Rad Laboratories, Hercules, CA). Caspases enzymatically cleave the substrate and release free amino-4-trifluoromethylcoumarin that produces a blue-green fluorescence, which is measured fluorometrically. Liver samples were homogenized in 100 µl of lysis buffer (10 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, and 20 µg/ml leupeptin), and hepatocytes were lysed in 50 µl of lysis buffer. Liver and cell lysates were prepared by four to five freeze and thaw cycles. Samples were centrifuged at 16,000 g in a microfuge for 10 min at 4°C. Supernatants (10 µl for hepatocyte extracts and 30 µl for liver extracts) were assayed for caspase activity by measuring fluorescence using a Perkin-Elmer luminescence spectrometer LS50B (Perkin-Elmer, Norwalk, CT) and normalized to protein using the Bradford assay (Bio-Rad).

Flow cytometric analysis. Primary rat hepatocytes (4.0 × 106 cells/plate) were plated in 100-mm tissue culture dishes. Cells were allowed to adhere to the plates in growth medium as described above. After 4 h of incubation, the cells were serum starved in HDM for 2 h and then were infected for 2 h with Ctrl or TGF-beta (A) adenoviruses. Cells were trypsinized and collected by centrifugation at 1,000 rpm for 5 min. Cell pellets were suspended in 300 µl of PBS, and 5 ml ice-cold 75% ethanol was added dropwise under gentle agitation to fix the cells. The cells were washed two times with PBS and were treated with 50 µg/ml RNase A in 1.12% sodium citrate at 37°C for 30 min. Cells were stained with 50 µg/ml propidium iodide in 1.12% sodium citrate overnight at 4°C, and samples were filtered through nitex membrane 3-60/45 (Sefar, Kansas City, MO). Samples were analyzed using a Becton-Dickinson Flow Cytometer (Becton-Dickinson, Fullerton, CA). Additionally, hepatocytes plated on 60-mm dishes were fixed with methanol-acetic acid (3:1) for 10 min at 4°C, washed two times with H2O, and dried. Cells were stained with propidium iodide (200 ng/µl) and were analyzed by ultraviolet fluorescence.

DNA ladder analysis. DNA was isolated from hepatocytes of Ctrl or TGF-beta (A)-infected cells 36 h after infection in the absence or presence of Z-VAD.FMK. Cells were scraped and suspended in 1 ml of HDM and lysed with 200 µl of 5× lysis buffer (2 M NaCl, 50 mM Tris · HCl, and 10 mM EDTA, pH 8.0), 50 µl 20% SDS, and 20 µg/ml proteinase K. Samples were incubated at 55°C for 45 min followed by the addition of 400 µl of 5 M NaCl. Samples were agitated well and centrifuged for 30 min at 3,500 rpm. The clear supernatant was removed, and 5 ml of 96% ethanol were added to precipitate the DNA. DNA was removed with a long-hooked Pasteur pipette and was washed in 1 ml of 70% ethanol. Samples were centrifuged at 16,000 g for 10 min at room temperature. The DNA was dried and suspended in 40 µl of Tris-EDTA containing 20 µg/ml RNase A and was incubated for 1 h at 37°C. Samples (50 µg) were run in a 1.5% agarose gel in 1× 0.04 M Tris-acetate-0.001 M EDTA running buffer.

Liver function analysis. Blood was obtained from animals at the time of death by direct inferior vena cava puncture and was serum stored at -80°C. The University of North Carolina Division of Laboratory Animal Medicine measured total bilirubin and alanine aminotransferase (ALT) concentrations from the serum, and growth media from infected hepatocytes with or without Z-VAD.FMK were also analyzed for lactate dehydrogenase (LDH) concentrations.

TGF-beta bioassay. A stably transfected fibroblast cell line harboring a TGF-beta response element controlling the expression of a luciferase reporter gene was seeded at a density of 2,000 cells/well in a 384-well microtiter plate. The medium consisted of DMEM plus 10% FCS (GIBCO-BRL), 2% (vol/vol) HEPES (GIBCO-BRL), and 1% (vol/vol) gentamicin (GIBCO-BRL). The total incubation volume was 1,000 µl/well. Cells were incubated for 1 day at 37°C and 5% CO2.

For TGF-beta determinations, the growth medium was removed, and 12.5 µl fresh medium (see above) plus 12.5 µl of the sera were added to each well and further incubated for 4 h. Subsequently, the medium was removed, the cells were lysed, and luciferase activity was measured. The relative luciferase signal was compared with the signals obtained with serial dilutions of TGF-beta 1 serving as a standard.

The half-maximal reporter signal was observed with TGF-beta 1 concentrations of ~20 pg/ml. The assay detects only TGF-beta (A) and does not detect TGF-beta (L). It does not discriminate between TGF-beta 1, TGF-beta 2, and TGF-beta 3. The assay also detects activin but with sensitivity about three orders of magnitude lower than for TGF-beta .

Data analysis. Data are presented as means ± SE.


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In vivo expression of TGF-beta 1. After injection of the adenoviruses, expression of the transgenes was confirmed by beta -galactosidase or luciferase activity or Northern blot analysis for TGF-beta 1 mRNA. Expression of beta -galactosidase or luciferase was demonstrated in all animals by increased enzyme activity in Ctrl-injected rats compared with TGF-beta 1-injected rats (data not shown). Twenty-four hours after injection, without PH, low levels of TGF-beta 1 mRNA were observed at 6, 12, and 18 h in TGF-beta (A) animals, but a dramatic increase was seen at 24 and 48 h in the rats injected with TGF-beta (L) or TGF-beta (A) (Fig. 1A). Translation of activated TGF-beta protein was confirmed by TGF-beta bioassay, which demonstrated an 8- to 10-fold increase in activated TGF-beta in TGF-beta (A)-injected rats compared with Ctrl- and TGF-beta (L)-injected rats (Fig. 1B). No animals (0 of 7) injected with TGF-beta (A) survived 24 h after PH; however, all animals survived 4 h after PH. Even a dose reduction to 1 × 109 PFU TGF-beta (A) was lethal in animals 24 h after PH. The survival rate of Ctrl- and TGF-beta (L)-injected rats was 100% after PH.


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Fig. 1.   Northern blot demonstrating time-dependent expression of transforming growth factor (TGF)-beta 1 in rat livers. A: lanes 1-10, RNA samples from animals injected with control (Ctrl), latent TGF-beta [TGF-beta (L)]-, or active TGF-beta [TGF-beta (A)]- containing adenoviruses. Total RNA was harvested from TGF-beta (L)-injected animals at 24 (lane 3) and 48 (lane 4) h and from TGF-beta (A)-treated animals at 6 (lane 5), 12 (lane 6), 18 (lane 7), 24 (lane 8), and 48 (lane 9) h after injections. Lanes 1 and 2 demonstrate RNA isolated from Ctrl-treated rats 24 and 48 h after injection, respectively. Additionally, lane 4 shows TGF-beta expression 24 h after partial hepatectomy (PH), and lane 10 shows expression 4 h after PH from either TGF-beta (L)- or TGF-beta (A)-injected livers, respectively. Equal loading was determined by ethidium bromide staining of the formaldehyde gel (data not shown). B: presence of activated TGF-beta protein is shown as the percent bioactivity measured from the serum of saline-, Ctrl, TGF-beta (L), and TGF-beta (A)-treated animals. Error bars represent SE.

Functional assessment of TGF-beta 1 in vivo. TGF-beta 1 increases alpha 1(I) collagen mRNA in the liver (7). We measured alpha 1(I) collagen mRNA by ribonuclease protection assay as a surrogate marker for functionally active TGF-beta 1 protein derived from adenoviral expression. TGF-beta (A)-injected animals showed a 2.7 ± 0.36-fold increase in alpha 1(I) collagen mRNA compared with Ctrl-injected animals 48 h after injection (Fig. 2, A and B). TGF-beta (L)-treated animals also showed a 3.5 ± 1.0-fold increase in alpha 1(I) collagen mRNA. This result suggests that functionally TGF-beta (A) protein is being made in vivo and that enough TGF-beta (L) is converted to the active form to increase hepatic stellate cell production of alpha 1(I) collagen mRNA.


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Fig. 2.   RNase protection assay (RPA) demonstrating alpha 1(I) collagen mRNA expression in rat livers treated with Ctrl, TGF-beta (L), or TGF-beta (A). A: RPA shows increases in alpha 1(I) mRNA expression in TGF-beta (L)-injected animals 48 h after injection (lane 6) and in TGF-beta (A)-injected animals 48 (lane 7) h after injections compared with saline-treated (lanes 1 and 2) or Ctrl (lanes 3 and 4) animals. B: quantitation by PhosphorImager analysis shows increases in alpha 1(I) collagen mRNA in rats treated with TGF-beta (L) or TGF-beta (A) 48 h after injections compared with rats injected with Ctrl viruses. Samples were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Error bars represent SE.

Hepatocyte DNA synthesis. DNA synthesis was assessed by proliferating cell nuclear antigen (PCNA) staining in uninfected (saline), Ctrl, and TGF-beta (L)-injected livers. DNA synthesis could not be assessed in TGF-beta (A)-injected rats because no animals survived 24 h after PH. Saline-, Ctrl, and TGF-beta (L)-injected rats demonstrated 1.7 ± 0.3, 1.5 ± 0.2, and 4.3 ± 2.1% hepatocytes in the S phase before PH, respectively. Twenty-four hours after PH, however, saline-, Ctrl, and TGF-beta (L)-injected animals demonstrated 31.9 ± 1.0, 26.2 ± 2.0, and 26.8 ± 3.1% replicating hepatocytes, respectively.

Hepatocyte mitosis was determined by counting the number of mitotic figures per high-power field on H&E-stained sections obtained 24 h after PH. Before PH, the mitotic index was low (0.20 ± 0.1 to 0.65 ± 0.2%) in all animals studied. After PH, the percentage of mitotic figures ranged from 6.9 ± 2.2 to 13.0 ± 1.5%.

TGF-beta (A) induces apoptosis in hepatocytes. TUNEL assay of Ctrl and TGF-beta (L)-injected rat livers showed little evidence of apoptosis, but TGF-beta (A)-injected rat livers demonstrated apoptotic hepatocytes (Fig. 3, A and B). Rats that did not undergo PH showed 0.4 ± 0.2 and 10.5 ± 1.8% apoptotic hepatocytes in Ctrl and TGF-beta (A)-injected animals 24 h after injection, respectively. The percentage of apoptotic hepatocytes increased to 32.1 ± 9.1% in TGF-beta (A)-injected animals 48 h after injection (Fig. 3, A and B). Four hours after PH, the percentage of apoptotic cells was 0.25 ± 0.2% in Ctrl-injected animals compared with 14.1 ± 1.3% in TGF-beta (A)-injected animals. Therefore, these results indicate that expression of TGF-beta (A) induces significant apoptosis in the normal adult liver.


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Fig. 3.   Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained liver sections. A: liver sections demonstrating apoptotic rat hepatocytes after Ctrl injection at 24 (a) and 48 (b) h and TGF-beta (A) injection at 24 (d) and 48 (e) h. PH was performed 24 h after injection with harvest of remnant livers 4 h after PH in Ctrl (c) and TGF-beta (A)-treated (f) rats. B: percentage of TUNEL-stained hepatocytes. Error bars represent SE.

TGF-beta (A) increases caspase 3-like and caspase 8-like activity. Caspase 3-like and caspase 8-like activities were measured to assess apoptosis signaling in Ctrl- and TGF-beta (A)-injected animals. Caspase 3-like and caspase 8-like activities were increased in TGF-beta (A)-injected rats 24 h after vector administration compared with Ctrl (Fig. 4). PH and TGF-beta (A) further increased caspase 3-like and caspase 8-like activities with nearly a fivefold increase in caspase 3-like activity and a fourfold increase in caspase 8-like activity (Fig. 4).


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Fig. 4.   Caspase 3-like and caspase 8-like activity measured from liver extracts. Caspase 3 and caspase 8 assays were performed on liver tissue from Ctrl and TGF-beta (A)-injected rats, and caspase activity is expressed as nmol amino-4-trifluoromethylcoumarin (AFC) · min-1 · µg protein-1. Liver cell lysates were assayed 24 h after adenoviral injection with or without PH or 4 h after PH. Error bars represent SE.

Analysis of hepatic function and histological analysis. Serum obtained at the time of death was used to assess hepatic function by measuring total bilirubin and ALT concentrations. Twenty-four hours after vector administration, bilirubin concentrations increased in TGF-beta (A)-injected rats compared with Ctrl animals, and further increases in bilirubin in TGF-beta (A)-injected rats were seen 4 h after a PH (Fig. 5A). Liver enzyme analysis also demonstrated increases in ALT concentrations in TGF-beta (A)-injected rats compared with Ctrl animals, and PH again further increased ALT (Fig. 5B). Collectively, these findings suggest that adenoviral vector administration may result in slight cytopathic effects but that TGF-beta (A) and PH produce marked alterations in hepatic function.


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Fig. 5.   Total bilirubin (TBIL) and alanine aminotransferase (ALT) concentrations from serum. Serum samples obtained at the time of liver harvest were assessed for concentrations of bilirubin (A) and ALT (B) from saline-, Ctrl, TGF-beta (L)-, and TGF-beta (A)-injected rats. Twenty-four hours after injection, serum was obtained for measurements at times 0, 4, and 24 h after PH. Error bars represent SE.

Light microscopy of liver sections of Ctrl, TGF-beta (L)-, and TGF-beta (A)-injected animals demonstrated no evidence of necrosis. A substantial number of apoptotic bodies was seen in TGF-beta (A)-injected animals but not in the other groups. Examination of the spleen, pancreas, kidney, heart, lung, intestine, and brain of moribund TGF-beta (A) animals showed no evidence of inflammation, necrosis, or apoptosis.

Z-VAD.FMK blocks TGF-beta (A)-induced apoptosis in primary hepatocytes. Primary rat hepatocytes were isolated and infected with either TGF-beta (A) or Ctrl adenoviruses in the presence and absence of the pancaspase inhibitor Z-VAD.FMK. Hepatocytes stained with propidium iodide showed increased membrane blebbing and nuclear condensation, characteristic of apoptosis, in TGF-beta (A)-infected cells compared with Ctrl cells (Fig. 6A, left). The addition of Z-VAD.FMK blocked apoptosis in TGF-beta (A)-infected cells (Fig. 6A, bottom right). Similar results were also observed by FACS analysis. TGF-beta (A)-infected cells showed an increase in the percentage of apoptotic hepatocytes compared with Ctrl cells (18.3 ± 0.35 vs. 10.4 ± 0.45%), and the addition of Z-VAD.FMK inhibited TGF-beta -induced apoptosis (18.3 ± 0.35 vs. 4.9 ± 0.3%; Fig. 6B). FACS data also showed similar percentages of hepatocytes in each phase of the cell cycle between Ctrl and TGF-beta (A)-infected cells, suggesting that TGF-beta (A) is not inducing cell cycle arrest but rather apoptosis (data not shown). TGF-beta (A)-infected cells resulted in DNA ladder formation (Fig. 6C, lane 3), whereas Ctrl cells demonstrated no ladder (Fig. 6C, lane 1). The inhibitor decreased DNA ladder formation in the TGF-beta (A)-infected cells (Fig. 6C, lane 4) and decreased both caspase 3-like and caspase 8-like activities in TGF-beta (A)-infected cells (Fig. 6D). LDH concentrations in media from TGF-beta (A)-infected cells were also increased compared with Ctrl cells (1,065 ± 3 vs. 500 ± 38 U/l, respectively), and diminished LDH concentrations were observed with the caspase inhibitor (1,065 ± 3 vs. 458 ± 13 U/l; Fig. 6E). Similar results were observed with ALT concentrations (data not shown). ALT concentrations increased in TGF-beta (A)-infected cells compared with Ctrl cells (45 ± 0 vs. 30.5 ± 0.5, respectively), and the caspase inhibitor blocked TGF-beta (A) induction of ALT (45 ± 0 vs. 32 ± 1). Expression of TGF-beta (A) in hepatocytes was confirmed by Northern blots (data not shown).


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Fig. 6.   Z-VAD.FMK, a pan-caspase inhibitor, blocks TGF-beta (A)-induced apoptosis in primary rat hepatocytes. Primary rat hepatocytes were infected with Ctrl or TGF-beta (A) adenoviruses for 2 h. After infection, the cells were treated with (+) or without (-) 5 µM Z-VAD.FMK. A: cells were fixed and stained with propidium iodide 36 h after infection. Left: cells without the inhibitor; right: cells treated with the inhibitor. B: cells were trypsinized, fixed with 75% ethanol, and stained with propidium iodide 36 h after infection. The percentage of apoptotic hepatocytes was assessed by FACS analysis. C: DNA was isolated 36 h after infection and run in a 1.5% agarose gel to examine DNA ladder formation. Lanes 1 and 2, DNA samples from Ctrl-infected cells; lanes 3 and 4, samples from TGF-beta (A)-infected cells. Lanes 1 and 3 have no Z-VAD.FMK, and lanes 2 and 4 represent samples with the inhibitor. D: caspase 3 and caspase 8 assays were performed on cell extracts from Ctrl and TGF-beta (A)-infected cells 36 h after infection in the absence or the presence of Z-VAD.FMK. Caspase activity is expressed as nmol AFC · min-1 · µg protein-1. E: lactate dehydrogenase (LDH) was measured from Ctrl and TGF-beta (A)-infected cells 36 h after infection without or with Z-VAD.FMK. Error bars represent SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we investigated whether autocrine expression of TGF-beta (L) or TGF-beta (A) would control hepatocyte growth in normal and regenerating liver. We hypothesized that TGF-beta (A) would inhibit hepatocyte replication after PH by inducing apoptosis and that these effects would not be observed in animals treated with TGF-beta (L). Surprisingly, we demonstrated that TGF-beta (A) but not TGF-beta (L) induced significant apoptosis in the normal liver, independent of PH. This is the first such study to show that autocrine expression of bioactive TGF-beta can modulate hepatocyte survival in normal liver. In addition, TGF-beta (A) was lethal to rats that underwent PH after TGF-beta (A) administration. Lethality was likely due to hepatic failure from apoptosis-induced insufficient liver volume. Increased caspase 3-like and caspase 8-like activity confirmed the apoptotic mechanism of death. These findings, however, were not present in rats treated with an adenoviral vector expressing TGF-beta (L). These findings suggest that TGF-beta (A) exhibits potent growth control of normal and proliferating hepatocytes by inducing apoptosis and that the conversion of TGF-beta (L) to TGF-beta (A) is a rate-limiting step in TGF-beta 1 hepatocyte growth control.

The mechanism(s) through which TGF-beta regulates hepatocyte growth control may be either cell cycle arrest or apoptosis. Previous studies have demonstrated that TGF-beta induces cell cycle arrest (1, 18, 47) in the G1 phase by preventing the hyperphosphorylation of pRb, which arrests cells at the G1/S phase checkpoint (15, 28). Further work has demonstrated that TGF-beta alters expression of G1-associated cyclins (18), cyclin-dependent kinases (18), and cyclin-dependent kinase inhibitors (47). Alternatively, TGF-beta controls epithelial cell growth, specifically hepatocytes, by inducing apoptosis (2, 4, 8, 12, 13, 20, 21, 36-38). Our study is the first to show that expression of bioactive TGF-beta can induce apoptosis in the normal liver. Our data suggest that TGF-beta (A) infection resulted in apoptosis and not cell cycle arrest, since the caspase inhibitor Z-VAD.FMK blocked the apoptotic response seen in primary hepatocytes (Fig. 6, A-E). Additionally, FACS analysis showed little change in the phases of the cell cycle when cells were infected with TGF-beta (A). The signaling pathways through which TGF-beta induces apoptosis are not known, but Gressner et al. (20) showed that calpain inhibitors decreased TGF-beta -induced apoptosis. Others have suggested that the transcriptional factor DPC4 and the stress-activated protein kinase pathway may mediate TGF-beta -induced apoptotic cell death (2). We have shown that caspase 3-like and caspase 8-like activity is increased markedly after administration of TGF-beta (A). TGF-beta -induced apoptosis is associated with the activation of caspase 3 and a cytokine response modifier A (CrmA)-inhibitable caspase (possibly caspases 1 or 8) in Hep 3B cells (10). This study (10) also showed that inhibition by CrmA is specific for the apoptotic effect of TGF-beta because CrmA did not alter the expression of TGF-beta -regulated promoters of the plasminogen activator inhibitor or cyclin A genes. The authors hypothesize that the function of CrmA is independent of the anti-proliferative and extracellular matrix-inducing effects of TGF-beta . Additionally, treatment of rat hepatocytes with TGF-beta resulted in apoptosis and increased CPP32-like protease activity that preceded the onset of apoptosis (26). Additionally, treatment of FAO rat hepatoma cells with TGF-beta induced caspase 2, but not caspase 1, activity (11). These authors surmised that caspase activity might be stimuli specific. The mechanisms of caspase activation, caspases involved, and role of mitochondria in TGF-beta -induced apoptosis require further investigation.

TGF-beta 1 is an inhibitor of epithelial cell growth, yet no models of exogenously or endogenously produced TGF-beta 1 growth arrest have demonstrated the lethality present in this study. We showed that administration of TGF-beta 1 alone resulted in apoptosis but was not lethal. The combination of TGF-beta (A) and PH, however, was uniformly lethal by 24 h. Even dose reduction to TGF-beta (A) reliably caused mortality in this model. Death was likely related to hepatic failure from insufficient liver volume because 10% of the hepatocytes in the hepatic remnant were apoptotic. Russell et al. (40) showed that exogenous delivery of TGF-beta 1 at the time of or after PH decreased hepatocyte DNA synthesis but did not inhibit liver regeneration and had no apparent cytopathic effect. Furthermore, in transgenic mice expressing bioactive TGF-beta 1, PH was not lethal, but hepatocyte DNA synthesis was reduced compared with wild-type mice (6). The findings in the current study demonstrate that autocrine expression of bioactive TGF-beta 1 and PH significantly alters hepatocyte growth control, and lethality is due to marked hepatic dysfunction as seen by increases in total bilirubin and ALT. Interestingly, release of ALT is usually associated with necrosis and not apoptosis. This study, however, clearly demonstrates increases in ALT concentrations with TGF-beta (A) infection (Fig. 5B), and these increases can be blocked by a caspase inhibitor in primary rat hepatocytes (data not shown), suggesting that release of ALT is associated with apoptotic cell death in hepatocytes.

Growth control of the liver has been examined extensively, with most investigations focusing on inducers of hepatocyte proliferation and hepatic reconstitution. TGF-beta 1 is the most well-studied inhibitor of hepatocyte growth, yet reports of TGF-beta 1 as a potent inhibitor of hepatocyte growth and regeneration remain unconvincing. Although TGF-beta inhibits hepatocyte DNA synthesis, the liver regenerates completely within days (40). TGF-beta 1 transcripts are not present in hepatocytes in the normal quiescent liver (5, 9) but are present after PH when TGF-beta 1 expression increases from 1 to 7 days (5). TGF-beta 1 is secreted in the latent form (17, 29), and bioactive TGF-beta 1 is required for growth inhibition (19). The current study shows that autocrine expression of TGF-beta (L) delivered to hepatocytes by an adenoviral vector did not inhibit hepatocyte proliferation or liver regeneration after PH. These findings suggest that the activation of TGF-beta from a latent molecule to a bioactive epithelial growth inhibitor represents a critical step of cellular growth control. In vitro mechanisms of TGF-beta activation include heat, extremes of pH, plasmin (29), and reactive oxygen species (3). TSP-1 has been shown to bind to LAP and convert TGF-beta (L) to an active molecule (44, 45). Recently, the integrin alpha vbeta 6 has been shown to activate TGF-beta in pulmonary inflammation and fibrosis (33), but this mechanism is unlikely in the liver since little alpha vbeta 6 is expressed in the liver.

Although the TGF-beta (L) failed to produce growth control in normal or proliferating hepatocytes, alpha 1(I) collagen production was increased in response to TGF-beta (L). TGF-beta is a potent stimulus for hepatic fibrogenesis (7), and this study demonstrates that the concentration of TGF-beta required for alpha 1(I) collagen production is substantially lower than that required to induce apoptosis and below the level of detection of our TGF-beta bioassay. Our previous study confirmed that administration of TGF-beta (L) was effective in increasing hepatic stellate cell alpha 1(I) collagen production 15-fold over control virus-injected mice and attests to the sensitivity of TGF-beta -induced collagen production (24).

We showed that bioactive TGF-beta was lethal to rats that underwent hepatectomy, and histological examination revealed hepatic failure from massive apoptosis. We were, however, unable to address the effects of autocrine expression of small amounts of bioactive TGF-beta . Although we decreased the dose by 60%, TGF-beta remained lethal. Presumably, infection with a smaller dose of TGF-beta (A) would result in survival, but whether this dose would permit substantial hepatocyte infection and allow detection of the transgene is unknown.

We demonstrated that autocrine expression of bioactive TGF-beta induces marked apoptosis in the normal liver and is associated with death after PH. This study suggests that bioactive TGF-beta may have important hepatic growth regulatory functions in both the normal liver and after PH. Apoptosis, via caspase activation, appears to be the mechanism of hepatocyte growth control, and activation of TGF-beta (L) may be an important regulatory step in hepatocyte growth control.


    ACKNOWLEDGEMENTS

We thank Angela Glover and Ellen Hughes for assistance with the preparation of the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34987 and by funds from the Center for Gastrointestinal Biology and Disease, University of North Carolina-Chapel Hill.

Address for reprint requests and other correspondence: K. E. Behrns, Dept. of Surgery, CB no. 7210, Chapel Hill, NC 27599-7210 (E-mail: Kevin_Behrns{at}med.unc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 November 1999; accepted in final form 18 August 2000.


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Am J Physiol Gastrointest Liver Physiol 280(1):G139-G148
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