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Constitutive over-expression of the insulin receptor substrate-1 causes functional up-regulation of Fas receptor
Journal of Hepatology 38 (2003) 803–810 www.elsevier.com/locate/jhep
Constitutive over-expression of the insulin receptor substrate-1 causes functional up-regulation of Fas receptor Marcus Wiedmann1,2, Seishu Tamaki1,2, Rebecca Silberman1,2, Suzanne M. de la Monte1,2, Leslie Cousens1,2, Jack R. Wands1,2,* 1
Department of Medicine, Liver Research Center, Rhode Island Hospital, Brown University School of Medicine, 55 Claverick Street 4th floor, Providence, RI 02903, USA Department of Pathology, Liver Research Center, Rhode Island Hospital, Brown University School of Medicine, 55 Claverick Street 4th floor, Providence, RI 02903, USA
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Background/Aims: Insulin- and insulin growth factor-1 stimulated signaling through the insulin receptor substrate-1 (IRS-1) promotes hepatocellular proliferation and survival. IRS-1 over-expression in transgenic (Tg) mouse livers caused constitutive activation of Erk mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) resulting in significantly increased levels of DNA synthesis and larger hepatic masses relative to non-transgenic (non-Tg) littermates. However, the livers eventually ceased to grow but remained approximately 25% larger than nonTg livers. We hypothesized that this growth homeostasis was achieved by parallel activation of pro-apoptosis pathways. Methods: Since Fas-mediated apoptosis is a common mechanism of hepatocyte destruction, we investigated the potential role of Fas receptor as a regulator of hepatic mass in IRS-1 transgenic mice. Results: Significantly increased Fas-receptor levels were detected in the livers of IRS-1 Tg compared to non-Tg mice by Western blot analysis. Functional activation of Fas-receptor in IRS-1 Tg livers was demonstrated by increased hepatocellular apoptosis caused by intravenous injection of anti-Fas (Jo-2). Conclusions: These findings suggest that the increased growth caused by IRS-1 over-expression is balanced by constitutive activation of pro-death mechanisms. Failure of the IRS-1 Tg mice to develop liver cancer may be due to preservation of pro-growth, pro-death homeostasis mechanisms. q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Insulin receptor substrate-1; Tumor necrosis factor a; Fas
1. Introduction Growth factor stimulated signaling through insulin receptor substrate-1 (IRS-1) mediates hepatocyte growth. Insulin, insulin-like growth factor type 1 (IGF-1), and ligands such as interleukin 4 [1,2], interferons a, b, g, and v, growth hormone, leukemia inhibitory factor, and tumor necrosis factor [1,3 –7], bind to cell surface receptors to Received 20 August 2002; received in revised form 10 February 2003; accepted 20 February 2003 * Corresponding author. Tel.: þ 1-401-444-7441; fax: þ1-401-444-2939. E-mail address: [email protected] (J.R. Wands). Abbreviations: IRS-1, insulin receptor substrate-1; Tg, transgenic; IGF-1, insulin growth factor; GH, growth hormone; TNF, tumor necrosis factor; Grb2, growth factor receptor-bound protein 2; PI3K, phosphatidylinositol-3 kinase; MAPK, mitogen activated protein kinase; MAPKK, mitogen activated protein kinase kinase; HCC, hepatocellular carcinoma.
activate their corresponding receptor tyrosine kinases, which then phosphorylate tyrosine residues on IRS-1. Tyrosyl phosphorylated (TP) IRS-1 can interact with adaptor molecules such as the growth factor receptorbound protein 2 (Grb2) [8,9], Syp-phosphatase (SH-PTP2) [10], and the p85 subunit of phosphatidyl-inositol-3 kinase (PI3K) [11,12] leading to downstream signaling that mediates growth, survival, and metabolic function [2,13 – 18]. We generated a transgenic (Tg) mouse model in which the human IRS-1 (hIRS-1) cDNA was over-expressed under the control of a human albumin promoter to demonstrate the relevance of IRS-1-transmitted signals in hepatocellular growth in vivo [19]. Increased IRS-1 protein levels in adult Tg mouse livers resulted in constitutively increased levels of TP-IRS-1, hepatocellular DNA synthesis, and PI3K and Erk mitogen activated protein kinase (MAPK) activities. The mean liver mass in the Tg-IRS-1 group was increased by
0168-8278/03/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-8278(03)00117-X
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approximately 25% due to chronic activation of pro-growth mechanisms. However, at 3 months of age, the livers ceased to grow despite sustained IRS-1 over-expression, suggesting that pro-death mechanisms may be important for regulating organ size. Since apoptosis is a standard means to rid the body of unneeded cells, compensatory activation of proapoptotic pathways might stabilize liver mass in hIRS-1 Tg mice. Fas-mediated signaling is an important and wellcharacterized mechanism of hepatic apoptosis [20,21]. Cell death results from Fas ligand binding to its receptor, followed by sequential recruitment of a series of binding partners with attendant caspase activation [22]. Fas-induced cell death can be mediated through Bid, mitochondrial permeability transition, and cytochrome c release followed by caspase activation [22 – 32]. Therefore, Fas-induced cell death can be mitochondria-dependent or independent. The present study was designed to examine the role of Fasmediated hepatocyte death for regulating liver mass in hIRS-1 Tg mice.
2. Materials and methods 2.1. Transgenic mouse model Tg mice that over-expressed the hIRS-1 cDNA under the control of a human albumin promoter and non-Tg control littermates were generated and maintained as previously described [19]. Humane care was administered, and study protocols complied with the institutional guidelines. The presence of Tg-hIRS-1 was verified by polymerase chain reaction analysis of genomic DNA extracted using the DNeasye kit (Quiagen, Valencia, CA). Over-expression of hIRS-1 in Tg mouse livers was confirmed by Western blot analysis with densitometry [19]. Hepatocyte proliferation indices were measured by bromo-20 -deoxyuridine (BrdU) and [3H]thymidine incorporation assays [19]. Relative hepatic mass was calculated from the ratio of liver weight to body weight in each animal.
2.2. Identification of pro-apoptosis mechanisms Fas-mediated cell death can be associated with increased Fas receptor or Fas ligand expression. To determine the relevance of this apoptosis mechanism in our model, Western blot analysis and semi-quantitative reverse transcription (RT) polymerase chain reaction (PCR) analysis were used to measure Fas receptor and Fas ligand levels in the liver tissue from 3-month-old Tg and non-Tg control littermates. For Western blot analysis [19], the tissues were homogenized in five volumes of NP-40 lysis buffer and protein concentration was measured using the Biorad reagent (BioRad, USA). Samples containing 100 mg of protein were fractionated by SDSPAGE, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), blocked with 3% non-fat dry milk, and incubated overnight at 48C with primary antibodies to Fas receptor or Fas ligand diluted in TBST containing 1% BSA. As a negative control, blots were stripped and re-probed with antibodies to tumor necrosis factor (TNF)-a. Immunoreactivity was revealed with horseradish peroxidase conjugated secondary antibody, enhanced chemoluminescence reagents (Amersham, Arlington Heights, IL), and film autoradiography. Immunoractivity was quantified by densitometric analysis of non-saturated autoradiographs using N.I.H. Image software. RT-PCR analysis was used to measure Fas receptor, Fas ligand, and GAPDH (negative control) mRNA expression. Total RNA was extracted by the method of Chomczynski and Sacchi [33]. First-strand cDNA synthesis was performed with 1 mg of total RNA using the AMV Kit for RT-PCR (Roche, Indianapolis, IN). Ten percent of cDNA products were used for PCR amplification in 50 ml reactions containing 1£ Taq polymerase buffer
(Roche, Indianapolis, IN), 50 pmol each of the upstream and downstream primers, 0.2 mM of dNTP, 2.5 mCi [a-32P]dCTP (DuPont NEN, Boston, MA), and 2.5 U of Taq DNA. The primers used for PCR were as follows: (1) murine Fas: sense, 50 -ACTTCTACTGCGATTCTCCTGG-30 , nt 453– 474/antisense 50 -ACCTGCTAGTCATTTGACCCTC-30 , nt 1251–1230; (2) murine FasL: sense 50 -TATACTTCCGGGGTCAGTCTTG-30 , nt 794– 815/antisense 5‘-AAC GGCCTCTGTGAGGTAGTAA-30 nt 1478–1457; and (3) murine GAPDH: sense 50 -GGCCAAG GTCATCCATGACAA-30 nt 520– 540/antisense 50 -GAAGTCGCAGGAGACAACCTGG-30 , nt 899– 867. The PCR was performed using 30 s for denaturation at 948C, 1 min annealing at 558C, and 2 min for extension at 728C for a total of 40 (Fas, FasL)/30 (GAPDH) cycles. The labeled PCR products were fractionated in 8% non-denaturing polyacrylamide gels and detected by autoradiography.
2.3. Functional activation of Fas receptor To determine if the increased Fas receptor expression in Tg-hIRS-1 livers was functionally active, we conducted experiments in which ten Tg-hIRS-1þ/2 mice and ten non-Tg controls (10 weeks old) were injected intravenously with 2 mg of a purified hamster monoclonal agonistic antiFas antibody (Jo-2). As a negative control, five additional mice in each group were injected intravenously with purified hamster anti-KLH IgG-1 (PharMingen, Los Angeles, CA). Mice were sacrificed 24 h after inoculation to harvest blood and liver tissue. Serum alanine aminotransferase (ALT) was measured using a Hitachi, Type 7150 automatic analyzer. Liver tissue was fixed in Histofix (Histochoice, Amresco, CA) and embedded in paraffin for histopathological studies, immunohistochemical staining, and in situ analysis of apoptosis. Fresh liver tissue samples were snap-frozen and used for Western blot analysis.
2.4. Histopathological studies Paraffin-embedded tissue sections were de-waxed, hydrated through graded alcohol, and stained with hematoxylin and eosin dyes. Since histopathological changes were relatively uniform in multiple adjacent sections, representative tissue sections were graded with respect to the degree of apoptosis as follows: Grade 0-normal liver; Grade I-individual cell apoptosis with 0–1% involvement; Grade II-micro-foci of apoptosis affecting up to 20% of the liver; Grade III-large foci of apoptosis involving 20–50% of the liver; Grade IV-massive apoptosis with hemorrhage affecting 50–90% of the liver parenchyma. The specimens were evaluated under code with random re-review to confirm consistency in grading.
2.5. In situ demonstration of apoptosis Paraffin-embedded sections were used to demonstrate nuclear DNA nicking, fragmentation and pyknosis associated with apoptosis. Nuclear DNA damage was detected using the in situ terminal deoxynucleotide transferase (TUNEL) assay for nicked DNA [34]. The 30 ends of nicked DNA were labeled with fluorescein isothiocyanate (FITC)-conjugated dUTP and terminal deoxynucleotide transferase enzyme (Roche Biochemicals). Nuclear pyknosis and fragmentation were detected by Hoechst H33258 staining [35]. Sections were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and examined by fluorescent microscopy.
2.6. Immunohistochemical staining Histological liver sections were immunostained in Tg-hIRS-1 and non-Tg littermates. Adjacent sections were immunostained with antibodies to Fas receptor using the avidin-biotin horseradish peroxidase method [36]. Immunoreactivity was revealed using True Bluee peroxidase substrate (KPL, Gaithersburg, MD). Sections were counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA) and mounted with Permount (Fisher).
2.7. Statistical analysis Between group comparisons were made using the Mann–Whitney U-test or analysis of variance with Yates correction or Student’s t-test with SPSS 8.0 software.
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2.8. Source of reagents Rabbit polyclonal antibodies to IRS-1 and the p85 subunit of PI3K were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Fas monoclonal antibody (Jo-2) and KLH (purified hamster IgG lambda isotype) were obtained from PharMingen (Los Angeles, CA). Monoclonal antibodies to Fas receptor and Fas ligand were purchased from Transduction Laboratories (Lexington, KY). Anti-TNF receptor Type I (p55; CD120a) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
3. Results 3.1. Transgenic mouse model Western blot analysis revealed the over-expression of the expected 185-kDa IRS-1 protein in Tg-IRS-1 livers as compared to non-Tg littermates (Figs. 1A,B). Overexpression of hIRS-1 was associated with a , 25% increase in mean liver mass in 3- and 12-month old mice (Fig. 1C), corresponding with the previously published results [19]. 3.2. Increased pro-apoptosis gene expression with IRS-1 over-expression Western blot analysis demonstrated increased Fas receptor expression in Tg-IRS-1 livers (N ¼ 6) compared with non-Tg littermates (N ¼ 6) (Fig. 2A). Densitometric analysis of non-saturated autoradiographs revealed 2.5-fold higher levels of Fas receptor in the Tg-IRS-1 group (P ¼ 0:004) (Fig. 2B). Correspondingly, increased Fas receptor expression was detected in liver tissue sections by immunohistochemical staining (Figs. 3A,B). In contrast, the hepatic levels of Fas ligand, TNF-a receptor and p85 subunit of PI3K were similar in the two groups (Figs. 2A,C,D). The results obtained by Western blot analysis were confirmed using RT-PCR, in that the Tg-IRS-1 mouse liver exhibited selectively increased expression of Fas receptor (data not shown). In addition, we examined Fas-receptor expression in the liver as a function of age and found a parallel increase at 6 and 12 months in both Tg-IRS-1 and non-Tg littermates. However, the Tg-IRS had consistently higher levels of Fas receptor expression at all age groups studied (data not shown.) 3.3. Functional activation of Fas receptor in Tg-hIRS-1 livers To determine if the over-expressed Fas receptor was functionally active, ten Tg-hIRS-1 and ten non-Tg littermates were injected intravenously with 2 mg anti-Fas IgG. In addition, five Tg-hIRS-1 and five non-Tg littermates were injected with purified hamster IgG lambda isotype standard antibody (anti-KLH). Mice were sacrificed 24 h after injection for histologic evaluation (Figs. 3 and 4). Hematoxylin and eosin stained liver sections were graded on a scale from 0 to IV according to the extent of
Fig. 1. Over-expression of IRS-1 in Tg mouse livers is associated with increased hepatic mass. (A) Representative Western blot analysis of IRS-1 with Tg-IRS-1 and non-Tg littermates. As a negative control, the blots were reprobed with antibodies to the p85 subunit of PI3K. (B) The Western blot signals were analyzed by densitometry and the mean levels (6SD) of IRS-1 protein detected in six samples from each group are depicted in the graph. Statistical comparisons were made using the Student’s t-test (**P < 0.01). (C) Comparison of relative hepatic mass in Tg-IRS-1 and non-Tg mice at 3 and 12 months of age (N 5 6 each). Graph depicts the mean (6SD) percentages of body weight accounted for by liver mass. Between-group comparisons were made using Student’s t-test (*P < 0.05).
apoptosis as follows: Grade 0-normal histology; Grade Iindividual eosinophilic/apoptotic bodies associated with scant lympho-mononuclear cell infiltrates; Grade IIoccasional clusters of apoptotic bodies affecting less than 20% of the tissue cross-section; Grade III-zonal apoptosis affecting mainly periportal regions and involving 20– 50% of the liver; Grade IV-extensive apoptosis with hemorrhage
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Fig. 2. Over-expression of CD95R (Fas receptor) in livers from Tg-IRS-1 mice. (A) Western blot analysis of CD95R (Fas receptor), CD95L (Fas ligand), and TNF-a R (tumor necrosis factor alpha receptor), and the p85 subunit of PI3K (negative control) expression in livers of Tg-IRS-1 and nonTg littermates. (B –D) Densitometric analysis of the Western blot digital signals corresponding to CD95R (B); CD95L (C); and TNF-a R (D). Graphs depict the mean 6 SD of results obtained for each group. Statistical comparisons were made using Student’s t-tests.
affecting . 50% of the liver. In general, Grade IV apoptosis involved . 90% of the liver. Livers from Tg-hIRS-1 and non-Tg littermates injected with anti-KLH control IgG exhibited little or no apoptosis. Anti-Fas Jo-2 injection produced apoptosis in all Tg-hIRS-1 mice, and in 80% of the non-Tg littermates. However, the overall degrees of hepatic apoptosis were more severe in the Tg-hIRS-1 group in that 5 of 10 scored at the Grade III –IV level, compared with 1 of 10 in the non-Tg group (P ¼ 0:035) (Figs. 3C – F and 4). In addition, Tg-hIRS-1 mice injected with Jo-2 had significantly increased ALT levels relative to anti-Fas injected non-Tg littermates (P , 0:05) (data not shown). 3.4. In situ demonstration of apoptosis In situ apoptosis was detected using a fluorescence-based TUNEL assay. The sections were counterstained with Hoechst H33258 to label all nuclei. The percentages of TUNELþ nuclei present in six adjacent 200 £ microscopic fields were determined by fluorescence microscopy and image analysis. All slides were examined under code. Low
densities of TUNELþ nuclei were detected in livers with Grade 0 or I injury, whereas Grade III or IV apoptosis, which occurred mainly in Tg-hIRS-1 mice, was associated with relatively high densities of TUNELþ nuclei. In livers with Grades 0– I injury, TUNELþ nuclei or apoptotic bodies were either rare and scattered, or absent (Fig. 5B). In livers with histological Grades III or IV injury, numerous clusters and isolated cells had TUNELþ nuclei (Fig. 5A). The mean (^ SD) percentage of apoptotic cells in Jo-2 injected Tg-hIRS-1 mice was 45.6 ^ 12.4% (range 0.5 – 95%) versus 5.45 ^ 3.22% (range: 0.5– 30%) in the non-Tg littermates (P ¼ 0:01) (Fig. 5C). Liver histology was normal and TUNELþ nuclei were not detected in livers from control mice injected with the non-relevant isotypematched antibody (anti-KLH). Finally, we compared the baseline level of apoptosis in the liver at 3 months between Tg-IRS-1 and non-Tg littermates. There was a significant increase in the Tg-IRS-1 mice (P , 0:05). However, mice examined at older ages showed little to no difference, and this observation may reflect, in part, the low sensitivity of the TUNEL assay.
M. Wiedmann et al. / Journal of Hepatology 38 (2003) 803–810
Fig. 3. Increased CD95R expression and increased sensitivity to antiFas R-mediated apoptosis in livers of Tg-IRS-1 mice. (A, B) Immunohistochemical staining for CD95R (Fas receptor) expression in TghIRS-1 (B); and non-Tg (A) littermate livers (original magnification 2003). Immunoreactivity was revealed by avidin-biotin horseradish peroxidase method using the TrueBlue Peroxidase chromogen. Note the prominent membranous labeling in the Tg-hIRS-1 samples (B) relative to control (A). (C –F) Low (C, D); and high (E, F) magnification images of hematoxylin and eosin stained sections of liver from non-Tg (C, E); and Tg-hIRS-1 (D, F) mice injected with the Jo-2 anti-Fas antibody. The non-Tg littermates (C, E) show normal histology, while the Tg-hIRS-1 livers (D, F) exhibit Grade IV apoptosis with large areas of cell loss (D, arrows) accompanied by hemorrhage and numerous apoptotic bodies (F, arrows) (original magnification C and D 5 100 3 ; E and F 5 400 3 ).
Therefore, to bring-out the difference between Tg-IRS-1 and non-Tg mice required a challenge with the Jo-2 anti-Fas receptor monoclonal antibody.
4. Discussion Insulin- and IGF-1-stimulated signaling through IRS-1 promotes hepatocellular proliferation and survival by activation of the Erk MAPK and PI3K-Akt pathways. We generated an hIRS Tg mouse model to demonstrate the relevance of these signaling cascades in terms of hepatocellular growth in vivo. A previous liver study showed constitutive activation of PI3K and MAPK cascades accompanied by increased cell proliferation as measured by BrdU labeling and [3H] thymidine incorporation in livers of Tg-hIRS-1 relative to non-Tg littermates [19]. The continuous growth signaling caused selective enlargement of Tg mouse livers (þ 25%) both with respect to overall
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Fig. 4. Distribution profile of liver injury observed in hematoxylin and eosin stained tissue sections from Tg-hIRS-1 and non-Tg mice injected with Jo-2 anti-Fas antibody. Livers from mock-injected controls were simultaneously studied. The grading criteria are summarized in the box below. The graph depicts the grades assigned to individual samples and the bars correspond to the median grade assignments. Inter-group statistical comparisons were made using the Mann–Whitney U-test.
hepatic mass and as a percentage of total body weight. However, the livers ceased to further enlarge beyond that achieved at 3 months of age, and they manifested no evidence of hepatocellular transformation. As these transgenic mice age, however, BrdU incorporation as a reflection of hepatocyte proliferation, decreases (Leonhard Mohr: personal communication). These observations raised important questions regarding the regulatory mechanisms involved in organ size homeostasis. Because the Fas signaling pathway is known to play an important role in hepatic regulation, we examined this system in the Tg mouse model [20,21,37]. The present study demonstrates that the major mechanism compensating for enhanced hepatocellular proliferation in hIRS-1 Tg mice is increased apoptosis sensitivity. In this regard, it is noteworthy that increased Fas receptor expression occurs with aging in rats [38] which was confirmed in our study, and that Fas-deficient mice exhibit hepatomegaly, splenomegaly and lymphadenopathy [39,40]. Conversely, Fas engagement accelerates liver regeneration after partial hepatectomy similar to TNF-a [41]. The Fas system is also involved in graft rejection following liver transplantation [42] and pathological conditions leading to hepatocellular injury and increased turnover, i.e. chronic hepatitis B and C, alcoholic hepatitis, acute hepatic failure of different etiology, autoimmune hepatitis, Wilson’s disease, bile duct disease and hepatocellular carcinoma (HCC) [43 – 57]. Fas receptor upregulation was striking in Tg compared
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anti-Fas differed in the Tg-hIRS-1 and non-Tg groups, such that i.p. injections of 3 mg Jo-2 induced 80% Grade III– IV apoptosis and 40% mortality in Tg-hIRS-1, compared with 40% Grade III– IV apoptosis and 100% survival in the non-Tg group (data not shown). Subsequent intravenous injections were performed to ensure uniform distribution in the liver [61]. The results documented a biologically active Fas signaling system associated with significantly increased levels of Fas receptor in Tg-hIRS-1 mouse livers. Biologically active Fas receptor was manifested by the significantly higher grades of hepatocellular apoptosis, increased percentages of TUNELþ nuclei, and higher ALT serum levels in Tg-hIRS-1 compared with non-Tg littermates when injected with Jo-2. From these results we conclude that increased growth caused by IRS-1 over-expression is balanced by constitutive activation of pro-death mechanisms. Since constitutive IRS-1 gene over-expression may contribute to the development of HCC [62 –65], it is likely that deregulation of pro-apoptotic pathways could be critical for promoting cellular transformation. In this regard, failure of Tg-IRS-1 mice to develop HCC thus far may be due to preservation of pro-growth, pro-death homeostasis mechanisms.
Acknowledgements M. Wiedmann was supported in part by Grant BMBFLPD 9801-11 from the Deutsche Akademie der Naturforscher Leopoldina, Germany, and the Liver Research Center by N.I.H. grants CA-35711, AA-02666 and P20 RR-15578 (J. Wands). Fig. 5. TUNEL assay showing of increased apoptosis in livers of Tg-IRS-1 (A) relative to non-Tg (B) littermates injected with Jo-2. DNA damage was detected by end-labeling with FITC-conjugated dCTP and terminal deoxynucleotide transferase. (C) TUNEL1 nuclei in six adjacent 2003 microscopic fields were enumerated under code, and the mean percentages calculated for each group. Inter-group comparisons were made using the Student’s t-test (**P 5 0.01).
to non-Tg littermates, whereas Fas ligand was similarly expressed in both groups. RT-PCR confirmed Fas receptor and Fas ligand co-expression in the liver, a phenomenon recently described for non-lymphoid tissue by others [37,52]. In addition, there was an increase in baseline apoptosis in Tg-IRS-1 mice compared to non-Tg littermates. The TNF-a receptor, containing the same death induction mechanism as Fas [58,59], was expressed at similar levels in the Tg and non-Tg control livers, suggesting some degree of specificity regarding liver size homeostasis mechanisms. To determine if increased Fas receptor expression was biologically active, we injected Tg-hIRS-1 and non-Tg littermates with agonistic Jo-2 anti-Fas antibodies known to activate CD95R in the same way as CD95L [60]. Pilot studies demonstrated that the threshold sensitivity for
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Constitutive over-expression of the insulin receptor substrate-1 causes functional up-regulation of Fas receptor Marcus Wiedmann1,2, Seishu Tamaki1,2, Rebecca Silberman1,2, Suzanne M. de la Monte1,2, Leslie Cousens1,2, Jack R. Wands1,2,* 1
Department of Medicine, Liver Research Center, Rhode Island Hospital, Brown University School of Medicine, 55 Claverick Street 4th floor, Providence, RI 02903, USA Department of Pathology, Liver Research Center, Rhode Island Hospital, Brown University School of Medicine, 55 Claverick Street 4th floor, Providence, RI 02903, USA
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Background/Aims: Insulin- and insulin growth factor-1 stimulated signaling through the insulin receptor substrate-1 (IRS-1) promotes hepatocellular proliferation and survival. IRS-1 over-expression in transgenic (Tg) mouse livers caused constitutive activation of Erk mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) resulting in significantly increased levels of DNA synthesis and larger hepatic masses relative to non-transgenic (non-Tg) littermates. However, the livers eventually ceased to grow but remained approximately 25% larger than nonTg livers. We hypothesized that this growth homeostasis was achieved by parallel activation of pro-apoptosis pathways. Methods: Since Fas-mediated apoptosis is a common mechanism of hepatocyte destruction, we investigated the potential role of Fas receptor as a regulator of hepatic mass in IRS-1 transgenic mice. Results: Significantly increased Fas-receptor levels were detected in the livers of IRS-1 Tg compared to non-Tg mice by Western blot analysis. Functional activation of Fas-receptor in IRS-1 Tg livers was demonstrated by increased hepatocellular apoptosis caused by intravenous injection of anti-Fas (Jo-2). Conclusions: These findings suggest that the increased growth caused by IRS-1 over-expression is balanced by constitutive activation of pro-death mechanisms. Failure of the IRS-1 Tg mice to develop liver cancer may be due to preservation of pro-growth, pro-death homeostasis mechanisms. q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Insulin receptor substrate-1; Tumor necrosis factor a; Fas
1. Introduction Growth factor stimulated signaling through insulin receptor substrate-1 (IRS-1) mediates hepatocyte growth. Insulin, insulin-like growth factor type 1 (IGF-1), and ligands such as interleukin 4 [1,2], interferons a, b, g, and v, growth hormone, leukemia inhibitory factor, and tumor necrosis factor [1,3 –7], bind to cell surface receptors to Received 20 August 2002; received in revised form 10 February 2003; accepted 20 February 2003 * Corresponding author. Tel.: þ 1-401-444-7441; fax: þ1-401-444-2939. E-mail address: [email protected] (J.R. Wands). Abbreviations: IRS-1, insulin receptor substrate-1; Tg, transgenic; IGF-1, insulin growth factor; GH, growth hormone; TNF, tumor necrosis factor; Grb2, growth factor receptor-bound protein 2; PI3K, phosphatidylinositol-3 kinase; MAPK, mitogen activated protein kinase; MAPKK, mitogen activated protein kinase kinase; HCC, hepatocellular carcinoma.
activate their corresponding receptor tyrosine kinases, which then phosphorylate tyrosine residues on IRS-1. Tyrosyl phosphorylated (TP) IRS-1 can interact with adaptor molecules such as the growth factor receptorbound protein 2 (Grb2) [8,9], Syp-phosphatase (SH-PTP2) [10], and the p85 subunit of phosphatidyl-inositol-3 kinase (PI3K) [11,12] leading to downstream signaling that mediates growth, survival, and metabolic function [2,13 – 18]. We generated a transgenic (Tg) mouse model in which the human IRS-1 (hIRS-1) cDNA was over-expressed under the control of a human albumin promoter to demonstrate the relevance of IRS-1-transmitted signals in hepatocellular growth in vivo [19]. Increased IRS-1 protein levels in adult Tg mouse livers resulted in constitutively increased levels of TP-IRS-1, hepatocellular DNA synthesis, and PI3K and Erk mitogen activated protein kinase (MAPK) activities. The mean liver mass in the Tg-IRS-1 group was increased by
0168-8278/03/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-8278(03)00117-X
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approximately 25% due to chronic activation of pro-growth mechanisms. However, at 3 months of age, the livers ceased to grow despite sustained IRS-1 over-expression, suggesting that pro-death mechanisms may be important for regulating organ size. Since apoptosis is a standard means to rid the body of unneeded cells, compensatory activation of proapoptotic pathways might stabilize liver mass in hIRS-1 Tg mice. Fas-mediated signaling is an important and wellcharacterized mechanism of hepatic apoptosis [20,21]. Cell death results from Fas ligand binding to its receptor, followed by sequential recruitment of a series of binding partners with attendant caspase activation [22]. Fas-induced cell death can be mediated through Bid, mitochondrial permeability transition, and cytochrome c release followed by caspase activation [22 – 32]. Therefore, Fas-induced cell death can be mitochondria-dependent or independent. The present study was designed to examine the role of Fasmediated hepatocyte death for regulating liver mass in hIRS-1 Tg mice.
2. Materials and methods 2.1. Transgenic mouse model Tg mice that over-expressed the hIRS-1 cDNA under the control of a human albumin promoter and non-Tg control littermates were generated and maintained as previously described [19]. Humane care was administered, and study protocols complied with the institutional guidelines. The presence of Tg-hIRS-1 was verified by polymerase chain reaction analysis of genomic DNA extracted using the DNeasye kit (Quiagen, Valencia, CA). Over-expression of hIRS-1 in Tg mouse livers was confirmed by Western blot analysis with densitometry [19]. Hepatocyte proliferation indices were measured by bromo-20 -deoxyuridine (BrdU) and [3H]thymidine incorporation assays [19]. Relative hepatic mass was calculated from the ratio of liver weight to body weight in each animal.
2.2. Identification of pro-apoptosis mechanisms Fas-mediated cell death can be associated with increased Fas receptor or Fas ligand expression. To determine the relevance of this apoptosis mechanism in our model, Western blot analysis and semi-quantitative reverse transcription (RT) polymerase chain reaction (PCR) analysis were used to measure Fas receptor and Fas ligand levels in the liver tissue from 3-month-old Tg and non-Tg control littermates. For Western blot analysis [19], the tissues were homogenized in five volumes of NP-40 lysis buffer and protein concentration was measured using the Biorad reagent (BioRad, USA). Samples containing 100 mg of protein were fractionated by SDSPAGE, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), blocked with 3% non-fat dry milk, and incubated overnight at 48C with primary antibodies to Fas receptor or Fas ligand diluted in TBST containing 1% BSA. As a negative control, blots were stripped and re-probed with antibodies to tumor necrosis factor (TNF)-a. Immunoreactivity was revealed with horseradish peroxidase conjugated secondary antibody, enhanced chemoluminescence reagents (Amersham, Arlington Heights, IL), and film autoradiography. Immunoractivity was quantified by densitometric analysis of non-saturated autoradiographs using N.I.H. Image software. RT-PCR analysis was used to measure Fas receptor, Fas ligand, and GAPDH (negative control) mRNA expression. Total RNA was extracted by the method of Chomczynski and Sacchi [33]. First-strand cDNA synthesis was performed with 1 mg of total RNA using the AMV Kit for RT-PCR (Roche, Indianapolis, IN). Ten percent of cDNA products were used for PCR amplification in 50 ml reactions containing 1£ Taq polymerase buffer
(Roche, Indianapolis, IN), 50 pmol each of the upstream and downstream primers, 0.2 mM of dNTP, 2.5 mCi [a-32P]dCTP (DuPont NEN, Boston, MA), and 2.5 U of Taq DNA. The primers used for PCR were as follows: (1) murine Fas: sense, 50 -ACTTCTACTGCGATTCTCCTGG-30 , nt 453– 474/antisense 50 -ACCTGCTAGTCATTTGACCCTC-30 , nt 1251–1230; (2) murine FasL: sense 50 -TATACTTCCGGGGTCAGTCTTG-30 , nt 794– 815/antisense 5‘-AAC GGCCTCTGTGAGGTAGTAA-30 nt 1478–1457; and (3) murine GAPDH: sense 50 -GGCCAAG GTCATCCATGACAA-30 nt 520– 540/antisense 50 -GAAGTCGCAGGAGACAACCTGG-30 , nt 899– 867. The PCR was performed using 30 s for denaturation at 948C, 1 min annealing at 558C, and 2 min for extension at 728C for a total of 40 (Fas, FasL)/30 (GAPDH) cycles. The labeled PCR products were fractionated in 8% non-denaturing polyacrylamide gels and detected by autoradiography.
2.3. Functional activation of Fas receptor To determine if the increased Fas receptor expression in Tg-hIRS-1 livers was functionally active, we conducted experiments in which ten Tg-hIRS-1þ/2 mice and ten non-Tg controls (10 weeks old) were injected intravenously with 2 mg of a purified hamster monoclonal agonistic antiFas antibody (Jo-2). As a negative control, five additional mice in each group were injected intravenously with purified hamster anti-KLH IgG-1 (PharMingen, Los Angeles, CA). Mice were sacrificed 24 h after inoculation to harvest blood and liver tissue. Serum alanine aminotransferase (ALT) was measured using a Hitachi, Type 7150 automatic analyzer. Liver tissue was fixed in Histofix (Histochoice, Amresco, CA) and embedded in paraffin for histopathological studies, immunohistochemical staining, and in situ analysis of apoptosis. Fresh liver tissue samples were snap-frozen and used for Western blot analysis.
2.4. Histopathological studies Paraffin-embedded tissue sections were de-waxed, hydrated through graded alcohol, and stained with hematoxylin and eosin dyes. Since histopathological changes were relatively uniform in multiple adjacent sections, representative tissue sections were graded with respect to the degree of apoptosis as follows: Grade 0-normal liver; Grade I-individual cell apoptosis with 0–1% involvement; Grade II-micro-foci of apoptosis affecting up to 20% of the liver; Grade III-large foci of apoptosis involving 20–50% of the liver; Grade IV-massive apoptosis with hemorrhage affecting 50–90% of the liver parenchyma. The specimens were evaluated under code with random re-review to confirm consistency in grading.
2.5. In situ demonstration of apoptosis Paraffin-embedded sections were used to demonstrate nuclear DNA nicking, fragmentation and pyknosis associated with apoptosis. Nuclear DNA damage was detected using the in situ terminal deoxynucleotide transferase (TUNEL) assay for nicked DNA [34]. The 30 ends of nicked DNA were labeled with fluorescein isothiocyanate (FITC)-conjugated dUTP and terminal deoxynucleotide transferase enzyme (Roche Biochemicals). Nuclear pyknosis and fragmentation were detected by Hoechst H33258 staining [35]. Sections were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and examined by fluorescent microscopy.
2.6. Immunohistochemical staining Histological liver sections were immunostained in Tg-hIRS-1 and non-Tg littermates. Adjacent sections were immunostained with antibodies to Fas receptor using the avidin-biotin horseradish peroxidase method [36]. Immunoreactivity was revealed using True Bluee peroxidase substrate (KPL, Gaithersburg, MD). Sections were counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA) and mounted with Permount (Fisher).
2.7. Statistical analysis Between group comparisons were made using the Mann–Whitney U-test or analysis of variance with Yates correction or Student’s t-test with SPSS 8.0 software.
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2.8. Source of reagents Rabbit polyclonal antibodies to IRS-1 and the p85 subunit of PI3K were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Fas monoclonal antibody (Jo-2) and KLH (purified hamster IgG lambda isotype) were obtained from PharMingen (Los Angeles, CA). Monoclonal antibodies to Fas receptor and Fas ligand were purchased from Transduction Laboratories (Lexington, KY). Anti-TNF receptor Type I (p55; CD120a) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
3. Results 3.1. Transgenic mouse model Western blot analysis revealed the over-expression of the expected 185-kDa IRS-1 protein in Tg-IRS-1 livers as compared to non-Tg littermates (Figs. 1A,B). Overexpression of hIRS-1 was associated with a , 25% increase in mean liver mass in 3- and 12-month old mice (Fig. 1C), corresponding with the previously published results [19]. 3.2. Increased pro-apoptosis gene expression with IRS-1 over-expression Western blot analysis demonstrated increased Fas receptor expression in Tg-IRS-1 livers (N ¼ 6) compared with non-Tg littermates (N ¼ 6) (Fig. 2A). Densitometric analysis of non-saturated autoradiographs revealed 2.5-fold higher levels of Fas receptor in the Tg-IRS-1 group (P ¼ 0:004) (Fig. 2B). Correspondingly, increased Fas receptor expression was detected in liver tissue sections by immunohistochemical staining (Figs. 3A,B). In contrast, the hepatic levels of Fas ligand, TNF-a receptor and p85 subunit of PI3K were similar in the two groups (Figs. 2A,C,D). The results obtained by Western blot analysis were confirmed using RT-PCR, in that the Tg-IRS-1 mouse liver exhibited selectively increased expression of Fas receptor (data not shown). In addition, we examined Fas-receptor expression in the liver as a function of age and found a parallel increase at 6 and 12 months in both Tg-IRS-1 and non-Tg littermates. However, the Tg-IRS had consistently higher levels of Fas receptor expression at all age groups studied (data not shown.) 3.3. Functional activation of Fas receptor in Tg-hIRS-1 livers To determine if the over-expressed Fas receptor was functionally active, ten Tg-hIRS-1 and ten non-Tg littermates were injected intravenously with 2 mg anti-Fas IgG. In addition, five Tg-hIRS-1 and five non-Tg littermates were injected with purified hamster IgG lambda isotype standard antibody (anti-KLH). Mice were sacrificed 24 h after injection for histologic evaluation (Figs. 3 and 4). Hematoxylin and eosin stained liver sections were graded on a scale from 0 to IV according to the extent of
Fig. 1. Over-expression of IRS-1 in Tg mouse livers is associated with increased hepatic mass. (A) Representative Western blot analysis of IRS-1 with Tg-IRS-1 and non-Tg littermates. As a negative control, the blots were reprobed with antibodies to the p85 subunit of PI3K. (B) The Western blot signals were analyzed by densitometry and the mean levels (6SD) of IRS-1 protein detected in six samples from each group are depicted in the graph. Statistical comparisons were made using the Student’s t-test (**P < 0.01). (C) Comparison of relative hepatic mass in Tg-IRS-1 and non-Tg mice at 3 and 12 months of age (N 5 6 each). Graph depicts the mean (6SD) percentages of body weight accounted for by liver mass. Between-group comparisons were made using Student’s t-test (*P < 0.05).
apoptosis as follows: Grade 0-normal histology; Grade Iindividual eosinophilic/apoptotic bodies associated with scant lympho-mononuclear cell infiltrates; Grade IIoccasional clusters of apoptotic bodies affecting less than 20% of the tissue cross-section; Grade III-zonal apoptosis affecting mainly periportal regions and involving 20– 50% of the liver; Grade IV-extensive apoptosis with hemorrhage
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Fig. 2. Over-expression of CD95R (Fas receptor) in livers from Tg-IRS-1 mice. (A) Western blot analysis of CD95R (Fas receptor), CD95L (Fas ligand), and TNF-a R (tumor necrosis factor alpha receptor), and the p85 subunit of PI3K (negative control) expression in livers of Tg-IRS-1 and nonTg littermates. (B –D) Densitometric analysis of the Western blot digital signals corresponding to CD95R (B); CD95L (C); and TNF-a R (D). Graphs depict the mean 6 SD of results obtained for each group. Statistical comparisons were made using Student’s t-tests.
affecting . 50% of the liver. In general, Grade IV apoptosis involved . 90% of the liver. Livers from Tg-hIRS-1 and non-Tg littermates injected with anti-KLH control IgG exhibited little or no apoptosis. Anti-Fas Jo-2 injection produced apoptosis in all Tg-hIRS-1 mice, and in 80% of the non-Tg littermates. However, the overall degrees of hepatic apoptosis were more severe in the Tg-hIRS-1 group in that 5 of 10 scored at the Grade III –IV level, compared with 1 of 10 in the non-Tg group (P ¼ 0:035) (Figs. 3C – F and 4). In addition, Tg-hIRS-1 mice injected with Jo-2 had significantly increased ALT levels relative to anti-Fas injected non-Tg littermates (P , 0:05) (data not shown). 3.4. In situ demonstration of apoptosis In situ apoptosis was detected using a fluorescence-based TUNEL assay. The sections were counterstained with Hoechst H33258 to label all nuclei. The percentages of TUNELþ nuclei present in six adjacent 200 £ microscopic fields were determined by fluorescence microscopy and image analysis. All slides were examined under code. Low
densities of TUNELþ nuclei were detected in livers with Grade 0 or I injury, whereas Grade III or IV apoptosis, which occurred mainly in Tg-hIRS-1 mice, was associated with relatively high densities of TUNELþ nuclei. In livers with Grades 0– I injury, TUNELþ nuclei or apoptotic bodies were either rare and scattered, or absent (Fig. 5B). In livers with histological Grades III or IV injury, numerous clusters and isolated cells had TUNELþ nuclei (Fig. 5A). The mean (^ SD) percentage of apoptotic cells in Jo-2 injected Tg-hIRS-1 mice was 45.6 ^ 12.4% (range 0.5 – 95%) versus 5.45 ^ 3.22% (range: 0.5– 30%) in the non-Tg littermates (P ¼ 0:01) (Fig. 5C). Liver histology was normal and TUNELþ nuclei were not detected in livers from control mice injected with the non-relevant isotypematched antibody (anti-KLH). Finally, we compared the baseline level of apoptosis in the liver at 3 months between Tg-IRS-1 and non-Tg littermates. There was a significant increase in the Tg-IRS-1 mice (P , 0:05). However, mice examined at older ages showed little to no difference, and this observation may reflect, in part, the low sensitivity of the TUNEL assay.
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Fig. 3. Increased CD95R expression and increased sensitivity to antiFas R-mediated apoptosis in livers of Tg-IRS-1 mice. (A, B) Immunohistochemical staining for CD95R (Fas receptor) expression in TghIRS-1 (B); and non-Tg (A) littermate livers (original magnification 2003). Immunoreactivity was revealed by avidin-biotin horseradish peroxidase method using the TrueBlue Peroxidase chromogen. Note the prominent membranous labeling in the Tg-hIRS-1 samples (B) relative to control (A). (C –F) Low (C, D); and high (E, F) magnification images of hematoxylin and eosin stained sections of liver from non-Tg (C, E); and Tg-hIRS-1 (D, F) mice injected with the Jo-2 anti-Fas antibody. The non-Tg littermates (C, E) show normal histology, while the Tg-hIRS-1 livers (D, F) exhibit Grade IV apoptosis with large areas of cell loss (D, arrows) accompanied by hemorrhage and numerous apoptotic bodies (F, arrows) (original magnification C and D 5 100 3 ; E and F 5 400 3 ).
Therefore, to bring-out the difference between Tg-IRS-1 and non-Tg mice required a challenge with the Jo-2 anti-Fas receptor monoclonal antibody.
4. Discussion Insulin- and IGF-1-stimulated signaling through IRS-1 promotes hepatocellular proliferation and survival by activation of the Erk MAPK and PI3K-Akt pathways. We generated an hIRS Tg mouse model to demonstrate the relevance of these signaling cascades in terms of hepatocellular growth in vivo. A previous liver study showed constitutive activation of PI3K and MAPK cascades accompanied by increased cell proliferation as measured by BrdU labeling and [3H] thymidine incorporation in livers of Tg-hIRS-1 relative to non-Tg littermates [19]. The continuous growth signaling caused selective enlargement of Tg mouse livers (þ 25%) both with respect to overall
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Fig. 4. Distribution profile of liver injury observed in hematoxylin and eosin stained tissue sections from Tg-hIRS-1 and non-Tg mice injected with Jo-2 anti-Fas antibody. Livers from mock-injected controls were simultaneously studied. The grading criteria are summarized in the box below. The graph depicts the grades assigned to individual samples and the bars correspond to the median grade assignments. Inter-group statistical comparisons were made using the Mann–Whitney U-test.
hepatic mass and as a percentage of total body weight. However, the livers ceased to further enlarge beyond that achieved at 3 months of age, and they manifested no evidence of hepatocellular transformation. As these transgenic mice age, however, BrdU incorporation as a reflection of hepatocyte proliferation, decreases (Leonhard Mohr: personal communication). These observations raised important questions regarding the regulatory mechanisms involved in organ size homeostasis. Because the Fas signaling pathway is known to play an important role in hepatic regulation, we examined this system in the Tg mouse model [20,21,37]. The present study demonstrates that the major mechanism compensating for enhanced hepatocellular proliferation in hIRS-1 Tg mice is increased apoptosis sensitivity. In this regard, it is noteworthy that increased Fas receptor expression occurs with aging in rats [38] which was confirmed in our study, and that Fas-deficient mice exhibit hepatomegaly, splenomegaly and lymphadenopathy [39,40]. Conversely, Fas engagement accelerates liver regeneration after partial hepatectomy similar to TNF-a [41]. The Fas system is also involved in graft rejection following liver transplantation [42] and pathological conditions leading to hepatocellular injury and increased turnover, i.e. chronic hepatitis B and C, alcoholic hepatitis, acute hepatic failure of different etiology, autoimmune hepatitis, Wilson’s disease, bile duct disease and hepatocellular carcinoma (HCC) [43 – 57]. Fas receptor upregulation was striking in Tg compared
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anti-Fas differed in the Tg-hIRS-1 and non-Tg groups, such that i.p. injections of 3 mg Jo-2 induced 80% Grade III– IV apoptosis and 40% mortality in Tg-hIRS-1, compared with 40% Grade III– IV apoptosis and 100% survival in the non-Tg group (data not shown). Subsequent intravenous injections were performed to ensure uniform distribution in the liver [61]. The results documented a biologically active Fas signaling system associated with significantly increased levels of Fas receptor in Tg-hIRS-1 mouse livers. Biologically active Fas receptor was manifested by the significantly higher grades of hepatocellular apoptosis, increased percentages of TUNELþ nuclei, and higher ALT serum levels in Tg-hIRS-1 compared with non-Tg littermates when injected with Jo-2. From these results we conclude that increased growth caused by IRS-1 over-expression is balanced by constitutive activation of pro-death mechanisms. Since constitutive IRS-1 gene over-expression may contribute to the development of HCC [62 –65], it is likely that deregulation of pro-apoptotic pathways could be critical for promoting cellular transformation. In this regard, failure of Tg-IRS-1 mice to develop HCC thus far may be due to preservation of pro-growth, pro-death homeostasis mechanisms.
Acknowledgements M. Wiedmann was supported in part by Grant BMBFLPD 9801-11 from the Deutsche Akademie der Naturforscher Leopoldina, Germany, and the Liver Research Center by N.I.H. grants CA-35711, AA-02666 and P20 RR-15578 (J. Wands). Fig. 5. TUNEL assay showing of increased apoptosis in livers of Tg-IRS-1 (A) relative to non-Tg (B) littermates injected with Jo-2. DNA damage was detected by end-labeling with FITC-conjugated dCTP and terminal deoxynucleotide transferase. (C) TUNEL1 nuclei in six adjacent 2003 microscopic fields were enumerated under code, and the mean percentages calculated for each group. Inter-group comparisons were made using the Student’s t-test (**P 5 0.01).
to non-Tg littermates, whereas Fas ligand was similarly expressed in both groups. RT-PCR confirmed Fas receptor and Fas ligand co-expression in the liver, a phenomenon recently described for non-lymphoid tissue by others [37,52]. In addition, there was an increase in baseline apoptosis in Tg-IRS-1 mice compared to non-Tg littermates. The TNF-a receptor, containing the same death induction mechanism as Fas [58,59], was expressed at similar levels in the Tg and non-Tg control livers, suggesting some degree of specificity regarding liver size homeostasis mechanisms. To determine if increased Fas receptor expression was biologically active, we injected Tg-hIRS-1 and non-Tg littermates with agonistic Jo-2 anti-Fas antibodies known to activate CD95R in the same way as CD95L [60]. Pilot studies demonstrated that the threshold sensitivity for
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