Title: Expression of a dominant-negative mutant TGF-beta type II receptor in transgenic mice reveals essential roles for TGF-beta in regulation of growth and differentiation in the exocrine pancreas
Abstract: Article15 May 1997free access Expression of a dominant-negative mutant TGF-β type II receptor in transgenic mice reveals essential roles for TGF-β in regulation of growth and differentiation in the exocrine pancreas Erwin P. Böttinger Corresponding Author Erwin P. Böttinger Albert Einstein College of Medicine, Bronx, NY, 10461 USAE.P.Böttinger and J.L.Jakubczak contributed equally to this work Search for more papers by this author John L. Jakubczak John L. Jakubczak Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USAE.P.Böttinger and J.L.Jakubczak contributed equally to this work Search for more papers by this author Ian S.D. Roberts Ian S.D. Roberts Department of Pathological Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Michelle Mumy Michelle Mumy Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Philipp Hemmati Philipp Hemmati Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Kerri Bagnall Kerri Bagnall Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Glenn Merlino Glenn Merlino Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Lalage M. Wakefield Lalage M. Wakefield Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Erwin P. Böttinger Corresponding Author Erwin P. Böttinger Albert Einstein College of Medicine, Bronx, NY, 10461 USAE.P.Böttinger and J.L.Jakubczak contributed equally to this work Search for more papers by this author John L. Jakubczak John L. Jakubczak Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USAE.P.Böttinger and J.L.Jakubczak contributed equally to this work Search for more papers by this author Ian S.D. Roberts Ian S.D. Roberts Department of Pathological Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Michelle Mumy Michelle Mumy Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Philipp Hemmati Philipp Hemmati Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Kerri Bagnall Kerri Bagnall Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Glenn Merlino Glenn Merlino Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Lalage M. Wakefield Lalage M. Wakefield Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Author Information Erwin P. Böttinger 1, John L. Jakubczak3, Ian S.D. Roberts4, Michelle Mumy2, Philipp Hemmati2, Kerri Bagnall2, Glenn Merlino3 and Lalage M. Wakefield2 1Albert Einstein College of Medicine, Bronx, NY, 10461 USA 2Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA 3Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA 4Department of Pathological Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PT UK The EMBO Journal (1997)16:2621-2633https://doi.org/10.1093/emboj/16.10.2621 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor-β (TGF-β) signaling in select epithelial cells. The dominant-negative mutant type II TGF-β receptor blocked signaling by all three TGF-β isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-β family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-β negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up-regulation of TGF-β expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-β signaling provides insights into mechanisms whereby loss of TGF-β responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-βs with associated paracrine effects on stromal compartments. Introduction The transfoming growth factors-βs (TGF-β) are multifunctional cytokines which regulate cell growth, differentiation and function (Roberts and Sporn, 1990), and recent evidence suggests that they constitute part of an important tumor suppressor pathway (Markowitz and Roberts, 1996). One or more of the three mammalian TGF-βs is expressed in nearly every tissue in the body, implicating this as a widely used regulatory system throughout development and adulthood (Flanders et al., 1989; Millan et al., 1991). However, despite a wealth of data on the multitude of biological activities of TGF-βs in vitro, in most cases the precise roles played by the TGF-βs in a particular in vivo setting are not known. This is in part because TGF-β action is strongly contextual. Thus, the specific effect of TGF-β on a particular cell appears to be an integrated function of the target cell type, its differentiated state and its environmental context, particularly regarding the nature of the extracellular matrix and the activities of other cytokines (Nathan and Sporn, 1991). For example, while TGF-β is a potent inhibitor of the growth of keratinocytes in vitro, transgenic overexpression of TGF-β in the skin can lead to an unexpected stimulation of keratinocyte proliferation in the basal state, but results in growth inhibition in a hyperplastic setting, following treatment with the phorbol ester TPA (Cui et al., 1995). This demonstrates that the physiological roles of TGF-βs in a particular tissue in vivo may not be readily predictable from its in vitro effects on cells derived from that tissue. As one approach to the molecular dissection of the in vivo biology of TGF-β, we wished to generate animal models in which TGF-β function is experimentally compromised in select tissues. The type I and type II TGF-β receptors (TβRI and TβRII) are activated by ligand-dependent formation of hetero-oligomeric complexes, in which TβRII transphosphorylates and activates TβRI, thereby initiating the signal transduction cascade (Wrana et al., 1994). In order to eliminate the TGF-β response in target tissues, we have used transgenic overexpression of a dominant-negative mutant form of the TβRII (Brand et al., 1993; Chen et al., 1993), resulting in tissue-restricted functional inactivation of the TGF-β receptor complex. This approach of local functional inactivation of genes has attractive advantages when compared with germline null mutations generated by gene targeting. First, loss of function can be targeted to specific cells in selected organs with appropriate transcriptional control elements, and potential embryonic lethality can be circumvented. Because of the important roles of TGF-βs during embryonic development, null mutations in the TβRII gene are expected to result in embryonic lethality. Second, the problem of isoform redundancy, a potential issue with the three mammalian TGF-β isoforms, can be eliminated. Third, confounding systemic effects such as widespread inflammation observed in multiple organs of the TGF-β1 null mouse (Kulkarni et al., 1993) can be avoided. We report here the effects of functional inactivation of TGF-β in target tissues in mice with expression of a dominant-negative mutant TβRII (DNR) under control of a metallothionein 1 (MT1) promoter (Palmiter et al., 1993). The results show that TGF-β negatively controls growth of pancreatic acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. Furthermore, loss of TGF-β responsiveness in DNR-positive acinar cells results in an unexpected increase in expression of TGF-β1 and TGF-β3 in vivo in the exocrine pancreas. This is associated with classic correlates of TGF-β overexpression such as fibrosis, angiogenesis and macrophage infiltration. These results have implications for understanding the complex roles of the TGF-β system in tumorigenesis. Results Generation of DNR transgenic mice and analysis of expression The DNR transgene encodes the extracellular and transmembrane domains of TβRII and was expressed under control of a mouse MT1 promoter and MT locus control regions (LCRs) (Figure 1A) (Palmiter et al., 1993). We generated two lines of transgenic mice, line AM3 with three copies, and line BF1 with one copy per haploid genome, respectively, in the strain FVB/N. Northern blotting revealed DNR RNA expression in both lines. In line AM3, the highest zinc-induced levels of DNR RNA were observed in the pancreas, liver, colon and small intestine (Figure 1B). Kidney, stomach and salivary gland had substantially lower levels of expression (Figure 1B). DNR expression was consistently lower in tissues from line BF1 mice when compared with line AM3 tissues (Figure 1C). In liver and pancreas, substantial levels of DNR were expressed without zinc induction (Figure 1C). To show the cellular localization of DNR protein in situ, we stained sections of pancreas and liver with anti-human TβRII(1–28) antibody. Cell surface-associated expression of DNR was observed in acinar cells in the pancreas and hepatocytes in the liver (Figure 2A and C). DNR expression was heterogeneous in both organs and was highest at 2 months of age. Figure 1.Construction of the DNR transgene and transgene expression. (A) Schematic representation of the transgene. A 19.1 kb SalI DNA fragment containing DNR was released from pMT-LCR-DNR and used for microinjections. MT-1, mouse metallothionein I promoter; hGH, poly(A) region of human growth hormone gene; 10 kb 5′ LCR and 7 kb 3′ LCR, 10 kb EcoRI fragment of the 5′ LCR and 7 kb EcoRI fragment of the 3′ LCR of the mouse MT I and II gene locus. (B) Tissue distribution of DNR transgene expression (line AM3). RNA (20 μg) of tissues from 2-month-old control littermate (−) and transgenic mice (+) maintained on drinking water containing 25 mM ZnSO4 was hybridized with 32P-labeled human DNR cDNA. (C) RNA (20 μg) from line AM3 liver (lanes 1 and 2) and pancreas (lanes 3 and 4), as well as pancreas from non-transgenic FVB/N (lane 5) and transgenic line BF1 (lane 6), was hybridized with a DNR probe. DNR expression in line AM3 liver and pancreas in the absence (lanes 1 and 3), or presence (lanes 2 and 4) of ZnSO4 in the drinking water. Download figure Download PowerPoint Figure 2.Expression of DNR in pancreas and liver. Immunostaining with anti-TβRII (residues 1–28) antibody: (A) pancreas of 2-month-old transgenic AM3 mouse in the absence or presence (see insert) of blocking peptide; (B) pancreas from non-transgenic FVB/N littermate; (C) liver of line AM3 mouse, (D) liver of FVB/N littermate. Arrows denote acinar staining (A) and hepatocyte staining (C), respectively. All animals were maintained on drinking water containing 25 mM ZnSO4. (A–D) all ×40. Download figure Download PowerPoint Dominant-negative mutant function of DNR in hepatocytes and acinar cells Ligand binding and receptor complex formation. Primary hepatocytes and pancreatic acinar cells were prepared from either line AM3 or from non-transgenic FVB/N mice. Ligand binding by DNR was assessed by receptor affinity labeling with [125I]TGF-β1. Ligand–receptor complexes were undetectable in lysates of affinity-labeled non-transgenic FVB/N hepatocytes, indicating low levels of endogenous receptors (Figure 3A). In hepatocyte lysates from line AM3, a prominent complex of ∼40 kDa represented DNR bound to [125I]TGF-β1, as confirmed by immunoprecipitation of affinity-labeled lysates with anti-human TβRII(1–28) (Figure 3A). Analogous experiments showed similar results using primary acinar cell preparations (Figure 3B). Figure 3.Dominant-negative mutant function of DNR in primary hepatocytes and acinar cells. Primary cultures of hepatocytes or purified pancreatic acini were generated from 2-month-old transgenic line AM3 and non-transgenic FVB/N mice. (A) Affinity labeling with [125I]TGF-β1 of hepatocytes from FVB/N (lanes 1 and 2) and line AM3 mice (lanes 3 and 4), in the absence (lanes 1 and 3), or presence (lanes 2 and 4) of a 50-fold molar excess of unlabeled TGF-β1 (cold competitor). TGF-β receptor complexes are denoted. Lanes 5 and 6, immunoprecipitation of affinity-labeled lysates from line AM3 hepatocytes with control IgG (1.5 μg/ml) (lane 5) or with anti-TβRII(1–28) (1.5 μg/ml) (lane 6). (B) Immunoprecipitation with anti-TβRII(1–28) of affinity-labeled lysates from FVB/N (lane 1) or line AM3 (lane 2) acinar cells. TGF-β receptor complexes are denoted. (C) Growth inhibition as measured by [3H]thymidine incorporation after treatment with TGF-β1, -β2, -β3 or activin, as indicated, in acinar cells from non-transgenic FVB/N (black bars) or transgenic line AM3 mice (hatched bars). (D) Growth inhibition as measured by [3H]thymidine incorporation after treatment with TGF-β1, -β2 or -β3, as indicated, in primary hepatocytes from non-transgenic FVB/N (black bars) or transgenic line AM3 mice (hatched bars). (E) Effect of TGF-β1 on secretion of fibronectin by metabolically labeled primary hepatocytes from FVB/N or line AM3 mice (visualized by autoradiography). Band intensities were quantified by densitometry, and levels of expression are denoted as x-fold above baseline expression (1.0×) in untreated cells (lanes 1 and 4). Download figure Download PowerPoint Loss of TGF-β responsiveness. Primary pancreatic acinar cell cultures from control FVB/N mice showed a marked inhibition of [3H]thymidine incorporation when treated with either TGF-β1, TGF-β2, TGF-β3 or activin, a member of the TGF-β superfamily structurally closely related to TGF-β (Figure 3C). In contrast, TGF-β isoforms had no effect on [3H]thymidine incorporation in acinar cells from line AM3 (Figure 3C). However, activin inhibited [3H]thymidine incorporation in transgenic acinar cells (Figure 3C), indicating that DNR expression only inactivates TGF-β signaling, but not signaling by other members of the TGF-β superfamily. Primary hepatocytes from control FVB/N mice were growth inhibited by TGF-β isoforms (Figure 3D), but not by activin (data not shown). None of the three TGF-β isoforms inhibited growth of hepatocytes from line AM3 (Figure 3D). In addition, induction of fibronectin protein secretion by TGF-β1, as seen in FVB/N hepatocytes, was absent in line AM3 hepatocytes (Figure 3E). These results indicate that DNR expression in cells from transgenic mice completely abrogates both the growth inhibition mediated by the three TGF-β isoforms, and the TGF-β mediated induction of matrix-associated genes. Phenotypic characterization of transgenic DNR mice On macroscopic inspection, the pancreas of line AM3 mice of 5 months and older had a lighter color when compared with the pancreas from non-transgenic littermates. Relative pancreatic weight expressed as a percentage of total body weight was significantly decreased in line AM3 mice when compared with FVB/N mice at 8 months of age (0.82 ± 0.10% versus 1.08 ± 0.03%, P <0.05). All other organs including the liver, colon and small intestine were macroscopically normal. Histological analysis of the pancreas showed severe abnormalities, including ductular transformation, neoangiogenesis, inter- and intralobular fibrosis and adipose replacement of acini in the exocrine pancreas of both transgenic lines AM3 and BF1 (see Table I; Figures 4 and 5). The islets of Langerhans appeared normal, consistent with the absence of DNR expression in endocrine cells. The abnormalities in the exocrine pancreas were generally more severe and occurred at a younger age in line AM3 when compared with line BF1, consistent with increased levels of DNR expression in the AM3 pancreas (Figure 1C). We focused therefore on line AM3 for a detailed analysis of the phenotype in the pancreas. Figure 4.Phenotype of mouse pancreas. (A and B) (×40) H&E staining of sections of the pancreas in 6-week-old non-transgenic FVB/N (A) and transgenic line AM3 mice (B); the arrows depict aberrant ductular cells and ductules; arrowheads denote microvessels. (C and D) (×40) Anti-cytokeratin immunostaining for markers of ductal epithelial cells in the pancreas of 6-week-old control FVB/N (C) and line AM3 mice (D); the arrow shows staining of an aberrant ductule. (E and F) (×10) H&E staining of pancreas sections of 15-month-old FVB/N (E) and transgenic line AM3 mice (F); the arrow depicts adipose replacement of acini, arrowheads show ductular and tubular structures. (G) (×40) H&E staining of a pancreas section of a 14-month-old transgenic line AM3 mouse; the arrow depicts an acinus undergoing dedifferentiation into a tubular complex. Most acini in this lobule have dedifferentiated into tubular complexes (see arrowhead). (H) (×5) Low power view of H&E staining of a pancreas section of a 19-month-old line AM3 mouse showing an eosinophilic focus of acinar cells (oval structure). Download figure Download PowerPoint Figure 5.Stromal abnormalities in transgenic mouse pancreas. (A and B) (×40) Reticulin staining of pancreas sections of 8-month-old FVB/N (A) and line AM3 mice (B); the arrow shows collagen deposition. (C and D) (×40) Anti-von Willebrand factor immunostaining of pancreas sections of 3-month-old FVB/N (C) and line AM3 mice (D); the arrows depict aberrant microvessels. (E and F) (×40) Immunostaining for macrophages in pancreas sections of 3-month-old FVB/N (E) and line AM3 mice (F); the arrow denotes Mac-2-positive macrophages. Download figure Download PowerPoint Table 1. The phenotype of line AM3 Line AM3 Controls (FVB/N) Age (months) 0–4 5–8 9–14 14+ 0–4 5–8 9–14 14+ Total no. of mice 26a 21 12 33 14 8 12 11 Ductular structures (%) 100b 100 100 100 0 0 0 0 Fibrosis/angiogenesis (%) 69 100 100 100 0 0 0 0 Adipose replacement (%) 27 100 100 100 0 13 25 27 Tubular complexes (%) 12 24 33 55 0 0 0 9 a Total number of mice per age group. b Percentage of mice with pathologic lesions in this age group. The earliest feature of the phenotype was the appearance of aberrant non-acinar cells in the exocrine pancreas from 3 weeks of age. These cells increased progressively in number and formed primitive ductular structures (Table I; Figure 4B). Strong staining with AE1/AE3 antibody, a marker of ductal epithelium, was observed in most of the aberrant non-acinar cells, confirming their ductal cell phenotype (Figure 4D). In addition, acinar cells in line AM3 mice were progressively replaced by adipose cells (Table I; Figure 4F). Tubular complexes, reflecting acinoductular metaplasia (Bockman, 1981), were found in some younger line AM3 mice and appeared frequently in older transgenic mice, often transforming entire lobules (Table I; Figure 4G). Within the pancreas of transgenic lines AM3 and BF1, the severity of the described abnormalities correlated well with the level of DNR expression in affected lobules. Older animals (>14 months) were also examined for the presence of eosinophilic foci of acinar cells, considered a marker of focal hyperplasia in rats (Eustis et al., 1990). Eosinophilic foci were not observed in non-transgenic FVB/N mice (n = 12), but were found in 21% (7/33) (P = 0.001) of line AM3 pancreas (Figure 4H). To better define the abnormalities in the transgenic pancreas, we applied specific staining techniques. Inter- and intralobular fibrosis developed by 5 months of age in line AM3 mice, and increased progressively (Table I). Reticulin staining of pancreas sections for collagen demonstrated significant expansion of the extracellular matrix in 8-month-old line AM3 mice when compared with FVB/N mice (18.6 ± 5.8% versus 3.5 ± 0.5% reticulin-stained section surface, respectively; P <0.05) (Figure 5A and B). Consistent with this, RNA expression levels of collagen 1, fibronectin and tissue inhibitor of metalloproteinase 1 (TIMP-1) were increased in line AM3 pancreas when compared with non-transgenic pancreas at 1, 3, 6 and 10 months of age (data not shown). Increased angiogenesis (see also Figure 4B) and macrophage infiltration were revealed by immunostaining for von Willebrand factor VIII (Figure 5D) and Mac-2 antigens (Figure 5F), respectively. TGF-β regulates growth and affects differentiation of acinar cells in the exocrine pancreas in vivo Proliferation and apoptosis. To address possible mechanisms underlying the observed pancreatic phenotype, we analyzed proliferative and apoptotic rates in the pancreas. Immunohistochemistry for proliferating cell nuclear antigen (PCNA) demonstrated a large increase in the proportion of acinar cells in cell cycle in line AM3 pancreas when compared with control FVB/N pancreas at 6 weeks (Table II; Figure 6A and B) and 8 months of age (Table II). This was accompanied by large numbers of apoptotic cells in line AM3 pancreas, but not in FVB/N pancreas (Table II; Figure 6C and D). The aberrant ductular epithelial cells present in line AM3 pancreas showed no evidence of increased proliferative activity, as demonstrated by the low frequency of PCNA staining in these cells (1.2 ± 0.4% at 6 weeks of age), when compared with acinar cells (35.0 ± 5.1% at 6 weeks of age) (Figure 6A). These results indicated that inactivation of TGF-β signaling in acinar cells causes an increase in proliferation and apoptosis throughout adult life in transgenic mice. In contrast, rates of proliferation and apoptosis in the liver were not significantly different between line AM3 mice and FVB/N littermates (data not shown). Figure 6.Proliferation, apoptosis and differentiation in the transgenic pancreas. (A) PCNA immunostaining in the pancreas of 6-week-old transgenic line AM3 mice; the arrow denotes a PCNA-positive acinar cell nucleus; the arrowhead shows lack of PCNA staining in non-acinar cells (×40). (B) PCNA staining in non-transgenic FVB/N mice (×40). (C) H&E staining of a pancreas section of 6-week-old transgenic line AM3 mice; the arrows show apoptotic cell bodies (×40). (D) Control FVB/N mice (×40). (E) Toluidine blue staining of 0.5 μm semi-thin sections of the pancreas of 3-month-old transgenic line AM3 mice (×100); the arrow shows ductular cells containing zymogen granules and the arrowhead denotes a remnant acinar cell in the ductular structure. (F) Control FVB/N mice (X100). Download figure Download PowerPoint Table 2. Rates of proliferation and apoptosis in the exocrine pancreas of non-transgenic FVB/N and transgenic line AM3 mice 6 weeks nc 8 months n PCNAa Apoptosisb PCNA Apoptosis FVB/N 185 ± 93 0 ± 0 5 12.7 ± 4.5 0.7 ± 0.6 3 AM3 678 ± 258d 7 ± 2d 7 74.7 ± 27.3d 4.5 ± 1.2d 6 a PCNA-positive acinar cells/mm2. b Apoptotic cells/mm2. c Number of mice. d P < 0.05. Differentiation and dedifferentiation. The accumulation of largely non-proliferating ductular cells and duct-like structures with the loss of acini suggested that normal differentiation and/or maintenance of a differentiated phenotype were disturbed in the transgenic pancreas. Toluidine blue staining of semi-thin (0.5 μm) sections of line AM3 pancreas showed that acini were replaced by ductular structures harboring transitional cells, characterized by ductal morphology, but containing zymogen granules (Figure 6E). Within the same structure, cells with remnant acinar morphology were present (Figure 6E). Dedifferentiation of acini into ductal structures as so-called ‘tubular complexes’ has been described in association with neoplastic and inflammatory conditions in the pancreas (Bockman, 1981). Tubular complexes were present in severely affected pancreatic lobules in line AM3 mice with increasing age (Table I; Figure 4G). These findings suggest that inactivation of TGF-β signaling in acinar cells in vivo is associated with dedifferentiation of acini into duct-like structures and tubular complexes. In addition, ductal cells may accumulate in association with high rates of acinar cell turnover (see Table II) in the transgenic pancreas. Inactivation of TGF-β signaling results in a selective increase of expression of TGF-β isoforms Because increased expression of matrix-associated genes and angiogenesis in vivo have been associated with increased TGF-β activity (Roberts et al., 1986), we examined levels of expression of TGF-β isoforms. By Northern blot analysis, expression of TGF-β1 and TGF-β3 RNA were markedly increased in line AM3 pancreas, and TGF-β2 RNA levels were slightly increased (Figure 7A). Similar results were obtained at 1, 2, 6 and 10 months of age. When compared with primary acinar cells from FVB/N control littermates, primary acinar cells from line AM3 mice had increased levels of TGF-β1 RNA (Figure 7B). Immunostaining with TGF-β isoform-specific antibodies showed little staining for TGF-β1, TGF-β2 and TGF-β3 in the exocrine pancreas of FVB/N mice (Figure 8). However, TGF-β1 protein was markedly increased in acinar cells and aberrant non-acinar cells, and TGF-β3 protein was markedly increased exclusively in acinar cells in the pancreas of line AM3 (Figure 8). TGF-β2 staining was not changed (Figure 8). These results suggest that inactivation of TGF-β signaling in acinar cells in vivo leads to increased expression of TGF-β1 and TGF-β3 isoforms. Figure 7.Expression of TGF-β isoforms in the transgenic pancreas. (A) RNA (10 μg) from the pancreas of 3-month-old non-transgenic FVB/N (lane 1) and transgenic AM3 (lane 2) mice was hybridized with TGF-β1, TGF-β2 and TGF-β3 cDNA probes. Hybridization with a ribosomal 18S probe shows equal loading. (B) RNA (10 μg) from primary acinar cell cultures generated from control FVB/N (lane 1) and line AM3 mice (lane 2) hybridized with a TGF-β1 probe. Ethidium bromide-stained agarose gel demonstrating 28S and 18S ribosomal complexes. Download figure Download PowerPoint Figure 8.In situ expression of TGF-β isoforms. Immunostaining for TGF-β1 (A and D), TGF-β2 (B and E) and TGF-β3 (C and F) in pancreas sections of 6-month-old transgenic line AM3 mice (A–C) and non-transgenic FVB/N littermates (D–E) (all ×40). Arrows show staining for TGF-β1 (A) and TGF-β3 (C) in acini in lobules with histologic changes; the arrowhead in (A) depicts TGF-β1 staining in aberrant non-acinar cells. Download figure Download PowerPoint Discussion Using a dominant-negative mutant receptor approach in transgenic mice, we have demonstrated for the first time the feasibility of inactivating TGF-β in select tissues in the whole animal. Specifically, we have shown that TGF-βs play an essential role in maintaining epithelial homeostasis and the differentiated phenotype in the exocrine pancreas, as demonstrated by severely perturbed cellular proliferation parameters and development of characteristic histologic abnormalities in the exocrine pancreas of two transgenic lines. While hepatocytes and acinar cells derived from the transgenic mice showed no growth inhibition or gene induction in response to any of the three TGF-β isoforms, growth inhibition induced by activin, the TGF-β superfamily member most closely related to TGF-β, was not affected. This confirms that the dominant-negative effect of this construct is confined to TGF-β1, 2 and 3. Mechanisms underlying the disruption of homeostasis Our data clearly indicate that TGF-β is an essential negative regulator of pancreatic acinar cell growth in vivo, consistent with in vitro data (Logsdon et al., 1992). We observed no changes in hepatocyte proliferation, apoptosis or collagen deposition in vivo in the liver of DNR transgenic mice when compared with normal mice, despite total loss of responsiveness to TGF-β in DNR transgenic hepatocytes in vitro. This suggests that TGF-β may not be a direct inhibitor of hepatocyte proliferation in the normal liver, and that the relative contribution of endogenous TGF-βs to epithelial homeosta