Human insulin-like growth factor-IA expression in transgenic mice promotes adenomatous hyperplasia but not pulmonary fibrosis

Stephen K. Frankel,2,3 Billie M. Moats-Staats,6 Carlyne D. Cool,2,5 Murry W. Wynes,1,4 Alan D. Stiles,6 and David W. H. Riches1,3,4

1Program in Cell Biology, Department of Pediatrics, and 2Interstitial Lung Disease Program, Department of Medicine, National Jewish Medical and Research Center, and Division of Pulmonary Sciences and Critical Care Medicine, Departments of 3Medicine, 4Immunology, and 5Pathology, University of Colorado Health Sciences Center, Denver, Colorado; and 6Department of Pediatrics, Division of Neonatal-Perinatal Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Submitted 9 November 2004 ; accepted in final form 17 December 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin-like growth factor-I (IGF-I) has been implicated in postnatal alveolar development, pulmonary fibrosis, and non-small cell lung cancer. To further investigate the role of IGF-I, we created a line of transgenic mice in which alveolar type II epithelial cells express human IGF-IA under the control of the surfactant protein C promoter. We determined the effect of pulmonary overexpression of human IGF-IA on 1) pulmonary inflammation and fibrosis in response to intratracheal instillation of bleomycin, 2) premalignant pulmonary adenomatous hyperplasia, and 3) adenoma formation. Transgenic expression of human IGF-IA had no effect on baseline gross lung pathology, cellularity of bronchoalveolar lavage, or total lung collagen content. In addition, there were no significant differences between transgenic mice and nontransgenic littermate controls in the development of pulmonary inflammation or pulmonary fibrosis in response to intratracheal bleomycin instillation. However, pulmonary expression of human IGF-IA in older mice (>12 mo) significantly increased the incidence of premalignant adenomatous hyperplastic lesions compared with littermate controls without affecting adenoma formation. These findings suggest that increased expression of human IGF-IA in alveolar air spaces does not affect the development of pulmonary fibrosis but promotes premalignant changes in the alveolar epithelium.

lung; bleomycin


INSULIN-LIKE growth factor-I (IGF-I) is a 70-amino acid growth factor that regulates development, growth, repair, and aging through its actions on multiple tissues and cell types (17, 18). IGF-I exhibits pleiotropic activities that include the stimulation of cell proliferation and the protection of cells against apoptosis (26, 27). In addition, IGF-I has been shown to stimulate collagen matrix synthesis by lung fibroblasts (9) and thus may play a role in lung development and remodeling. The liver is the primary source of circulating IGF-I, although IGF-I is synthesized and secreted by most organs and tissues, including the lung. Extrahepatic expression of IGF-I is regulated by promoter 1 (12) and gives rise via alternative splicing to two forms of precursor IGF-I, namely, IGF-IA and IGF-IB, that differ in their COOH-terminal E peptides (30). Human IGF-IA contains a 35-amino acid E peptide, whereas the E peptide of IGF-IB comprises 77 residues. Both E peptides are removed during processing of precursor IGF-I to the mature 7.6-kDa protein.

IGF-I is expressed in the lung during normal postnatal alveolarization and is thought to contribute to the wave of mesenchymal and epithelial cell proliferation that accompanies alveolar septation (21). However, increased expression of IGF-I has also been detected in lung tissues in response to hyperoxia-induced lung injury and during the development of bleomycin-induced pulmonary fibrosis in mice (19, 37). In situ studies of lung biopsy specimens have shown increased expression of IGF-I by macrophages and alveolar epithelial cells in patients diagnosed with a variety of interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF), silicosis, asbestosis, and coal miner's pneumoconiosis, and in the fibroproliferative phase of acute respiratory distress syndrome (3, 15, 35, 36).

In addition to its effects on lung development and injury, IGF-I has been strongly implicated in the development and progression of tumors of the breast, colon, and pancreas as well as in non-small cell lung cancer (18, 38) through its ability to promote proliferation, adhesion, survival, and resistance to chemotherapy (18, 20, 38). Likewise, blockade of the IGF-I signaling cascade appears to represent a viable anticancer therapy for a number of adenocarcinomas, including non-small cell lung cancer (34). Most responses to IGF-I are induced by ligation of the IGF-I receptor, a receptor tyrosine kinase that stimulates signaling cascades involved in the regulation of the cell cycle and cell survival, including ERK and Akt (26, 27). Collectively, these findings suggest that IGF-I plays an important role not only in lung development but also in pulmonary injury, fibrosis, and carcinogenesis.

To further investigate the role of IGF-I in bleomycin-induced pulmonary fibrosis and inflammation and in lung cancer, we developed a line of transgenic mice in which human IGF-IA is overexpressed by alveolar type II epithelial cells under the control of the human surfactant protein C (SP-C) promoter. In this report, we show that transgenic overexpression of human IGF-IA in pulmonary epithelial lining fluid increased the incidence of premalignant epithelial adenomatous hyperplastic lesions in older mice but did not affect pulmonary inflammation or fibrosis either basally or in response to intratracheal instillation of bleomycin.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Recombinant human and mouse IGF-I were obtained from R&D Systems (Minneapolis, MN). Anti-phospho (Ser 473)-Akt antibody (no. 9277) and anti-Akt antibody (no. 9272) were purchased from Cell Signaling Technology (Beverly, MA). The anti-phospho-ERK antibody (no. V8031) was from Promega (Madison, WI), and the anti-ERK1/2 antibody (ERK1/2-CT, no. 06–182) was purchased from Upstate Biotechnology (Lake Placid, NY). Bleomycin (Bleocin) was generously donated by Bristol Squibb Myers (Princeton, NJ).

Construction of the fusion gene. A 358-bp fragment of a human IGF-IA cDNA (11) corresponding to bases 180–538 of the coding region was inserted into the plasmid pNeoNut (a gift from Dr. A. Joseph D'Ercole, University of North Carolina at Chapel Hill) between a rat somatostatin signal peptide (corresponding to bases 495–613) (10) and a polyadenylation sequence derived from the human growth hormone gene (7). The mouse metallothionein promoter sequences (25) of the plasmid pNeoNut were replaced with a 3.7-kb Sac1/Kpn1 human genomic fragment corresponding to the human SP-C promoter (13) (a gift from Dr. Jeffrey A. Whitsett, University of Cincinnati College of Medicine) to generate the plasmid SP-C/IGF-I, an IGF-IA expression plasmid under control of the surfactant-associated protein C promoter. The original pNeoNut plasmid driving IGF-I expression under control of the metallothionein promoter was previously shown to express IGF-IA in stably transfected Chinese hamster ovary cells (7). The SP-C/IGF-I plasmid was sequenced at the University of North Carolina Automated Sequence Facility using the dye terminator method (31) to ensure no mutations in the construct and the correct reading frame for translation.

Generation of transgenic mice. The human SP-C promoter/IGF-I fusion gene was excised from plasmid sequences by digestion with SacI and EcoRI restriction enzymes, isolated by gel electrophoresis in low melting temperature agarose (FMC Bioproducts, Rockland, ME), and purified from the agarose. The fusion gene was then precipitated with 2.5 vol of 95% ethanol at –20°C. After washing with 70% ethanol was completed, the DNA pellet was dissolved in TE buffer (10 mM Tris·HCl, pH 7.2, 1 mM EDTA). Microinjection of the fusion gene was performed at the Transgenic Mouse Facility of the Program in Molecular Biology and Biotechnology, University of North Carolina, using hybrid C3H/C57B6 fertilized mouse eggs.

Identification of transgenic mice. Mice expressing the human IGF-IA transgene were identified via PCR of the IGF-IA insert. Tail clips were obtained from each experimental mouse and digested overnight at 37°C with proteinase K in a shaking water bath. We then isolated genomic DNA using a DNA extraction kit as per the manufacturer's protocol (Qiagen, Valencia, CA). We amplified genomic DNA by PCR in a Perkin Elmer 24000 thermal cycler using these oligodeoxynucleotides: RSOMAT5, 5'-ATGCTGTCCTGCCGTCTCCA-3'M, corresponding to the rat somatostatin signal peptide bases 543 to 562 (10), and HIGFI3, 5'-CTGTCTCCACACACGAACTG-3' complementary to human IGF-I cDNA bases 222–241 (11). PCR conditions were as follows: 1 cycle of denaturation for 3 min at 95°C, annealing for 3 min at 55°C, and extension at 72°C for 5 min; this was followed by 36 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 55°C, and extension at 72°C for 3 min. PCR products were separated on a 2% agarose TAE gel. A band at 134 bp indicates a transgene-carrying animal, whereas no band indicates a wild type. All breeding pairs consisted of a heterozygous male and a wild-type female such that no homozygous transgenic animals were used. All animal breeding and experimental studies were approved by the institutional animal care and use committee at National Jewish Medical and Research Center.

Histology. Heart and lungs were isolated through careful dissection and removed from the euthanized animals en bloc with a 22-gauge catheter secured in the airway. Lungs were then fixed in 10% formalin under 25 cmH2O intra-alveolar pressure. Paraffin sections were then made at the National Jewish Medical and Research Center, Department of Pathology, Core Histology Facility, and stained with hematoxylin and eosin or pentachrome stain. All histological interpretations were performed by an expert lung pathologist (C. D. Cool) who was blinded to experimental conditions.

Western blotting. We detected specific proteins in cell lysates using Western blot analysis. Primary mouse lung fibroblasts were plated at 2.5 x 105 cells/well in six-well tissue culture plates and allowed to adhere overnight. Cells were washed in serum-free medium and allowed to quiesce for 30 min before stimulation with mouse IGF-I, human recombinant IGF-I, or serum-free medium alone. Media were removed at the specified time points, and 400 µl of lysis buffer [50 mM Tris·HCl, pH 7.4, 136 mM NaCl, 10% (vol/vol) glycerol, 1% NP-40, 1 mM NaF, 1 mM phenylmethysulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM Na3VO4] were added to each well of the six-well plate. The cells were scraped off the plate and processed as described (6).

Primary mouse lung fibroblast isolation. Primary cultures of mouse lung fibroblasts were derived from 8- to 12-wk-old male mice. Mice were euthanized by carbon dioxide asphyxiation, the lungs were removed in a sterile fashion, and the tissue was minced into 1- to 2-mm3 sections and placed into tissue culture in 10% FCS enriched tissue culture media on scored plates. After 5–10 days, pulmonary fibroblasts growing out of the tissue were passaged for routine tissue culture. All experiments were performed on early passage (passages 2–4) cells.

Bleomycin instillation. Mice between the ages of 8 and 12 wk were weighed, given an identification number, and instilled intra-tracheally with either bleomycin or an equivalent volume of saline. Tail clips were obtained, and the genotype of each mouse was subsequently determined (investigators were blinded to genotypic identity until data analysis). Mice were then euthanized at 14 or 28 days after instillation, and studies were performed as outlined below. All mice were anesthetized with 300 µl of intraperitoneally administered Avertin to unconsciousness. The skin overlying the trachea was cleaned with ethanol, and a single incision was made in the skin. Bleomycin (4 U/kg) at a concentration of 1.6 x 10–3 U/µl in sterile saline or an equivalent volume of saline was then instilled intratracheally after orotracheal intubation with a 22-gauge gavage. Gavage location was confirmed in each mouse by direct visualization. The wound was closed with Vetbond (3M, Minneapolis, MN), and the mouse was allowed to recover. Mice were euthanized with carbon dioxide asphyxiation at 14 and 28 days after instillation.

Bronchoalveolar lavage and cell counts. A 22-gauge catheter was introduced into the trachea of euthanized animals and secured with a single suture. Two 800-µl aliquots of 0.9% saline were then serially instilled and withdrawn. The pooled sample was centrifuged at 3,000 rpm in an Eppendorf benchtop centrifuge, and the supernatant was removed and stored at –80°C for quantification of IGF-I. The cell pellet was resuspended in DMEM. Total cell counts were determined by duplicate Coulter counter measurements of each sample. Cytospin preparations (500 rpm for 5 min) were fixed in methanol and stained with hematoxylin and eosin. Differential cell counts were performed by random field blinded counts of a total of 200 cells on duplicate samples.

Human IGF-I quantification. Bronchoalveolar lavage fluid (BALF) was thawed, and 500 µl of each sample were loaded into a Microcon YM-3 centrifugal filter device (Millipore, Bedford, MA). Samples were spun at 10,000 rpm in an Eppendorf benchtop centrifuge for 100 min. The concentrated samples were then assayed with a Quantikine human IGF-I immunoassay kit (R&D Systems) according to the manufacturer's instructions. Duplicate samples were performed for all mice, and the mean result is reported. No cross-reactivity with mouse IGF-I was detected with either recombinant or endogenous mouse IGF-I.

Hydroxyproline quantification. Total lung hydroxyproline levels were used as a well-validated marker of bleomycin-induced fibrosis (23). Both lungs were removed from an individual animal and snap frozen in a dry ice-ethanol bath and stored at –70°C. Samples were then lyophilized, and the dry lung weights were recorded. Lungs were ground to a fine powder with a mortar and pestle and hydrolyzed overnight with 6 N HCl at 115°C at a concentration of 20 µg/ml. Samples were filtered through glass wool and cooled. Five-microliter samples of hydrolyzed lung and hydroxyproline standards were plated in duplicate along with duplicate samples of 1:2 dilutions in 96-well plates and incubated for 20 min in 100 µl of 0.06 M chloramine T in citrate-acetate buffer, pH 6. Ehrlich's solution (100 µl; 1.2 M dimethylaminobenzaldehyde in 22% perchloric acid-n-propanol) was then added to each sample. After a 15-min incubation at 65°C, plates were analyzed in an ELISA plate reader at 550 nm for colorimetric analysis. Concentrations of each sample were determined by interpolation along a standard curve.

Statistical analysis. We performed all statistical analyses using GraphPad Prism version 3.0 software (GraphPad Software, San Diego, CA). Two-way ANOVA analysis was used to assess the effect of the IGF-IA transgene across experimental groups. Unpaired, two-tailed t-tests were used in comparisons made between individual experimental groups with numeric variables. A contingency table was used to compare the incidence of adenomatous hyperplasia, a binary variable.


    RESULTS
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Mouse lung fibroblasts respond to human IGF-I. Before conducting in vivo experiments using human IGF-I in a mouse model, we first determined whether mouse lung cells responded to human IGF-I with the same efficacy as mouse IGF-I. Primary cultures of mouse lung fibroblasts were stimulated with either recombinant human IGF-I, recombinant mouse IGF-I, or medium alone. The cells were then lysed and analyzed for activation of ERK and Akt signaling by SDS-PAGE and Western blot analysis using phospho-Akt and phospho-ERK antibodies. Both Akt and ERK were phosphorylated to a similar extent in response to human IGF-I and mouse IGF-I but not in response to medium alone (Fig. 1). Densitometry revealed a 7.7-fold increase in phospho-Akt relative to total Akt at 60 min after stimulation with mouse IGF-I (100 ng/ml) and a 10.1-fold increase in response to human IGF-I (100 ng/ml). Similarly, there was a 2.0-fold and a 2.5-fold increase in phospho-ERK relative to total ERK at 15 min in response to mouse and human IGF-I, respectively. Thus the activity of human IGF-I and mouse IGF-I with respect to mouse cells appears equivalent.



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Fig. 1. Human and mouse IGF-I (hIGF-I and mIGF-I, respectively) activate signaling responses in primary cultures of mouse lung fibroblasts. Mouse lung fibroblasts were incubated in media alone or were stimulated with recombinant hIGF-I or mIGF-I (both at 50 ng/ml) for 15 or 60 min to achieve maximal activation of ERK and Akt, respectively. Cells were lysed and analyzed by Western blot for activated phospho-Akt at 60 min and activated phospho-MAPK/ERK at 15 min. Immunoblots for Akt and ERK confirm the equality of protein loading. Results are representative of 3 separate experiments.

 
Human IGF-I is expressed in the lungs of the transgenic animals. Creating mice that express human IGF-IA (rather than mouse IGF-I) under the control of the human surfactant protein C promoter allowed us to investigate the consequences of pulmonary overexpression of IGF-I (14) and to distinguish between endogenous and transgenic IGF-I production. We initially determined whether the transgenic mice expressed human IGF-I protein in their alveolar air spaces as would be predicted by their genotype. Bronchoalveolar lavage was performed on transgenic mice and wild-type littermate controls, and the levels of human IGF-I in the BALF were determined by ELISA. Figure 2 shows that the transgenic animals secrete human IGF-I into their air spaces, whereas the wild-type animals exhibit no detectable human IGF-I (P < 0.0001).



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Fig. 2. hIGF-I is present in bronchoalveolar lavage fluid (BALF) from transgenic mice expressing hIGF-IA (IGF-ITG/–) but not in wild-type littermate controls. Bronchoalveolar lavage was conducted on euthanized mice, and hIGF-I was detected by ELISA.

 
Effect of transgenic expression of human IGF-IA on lung histology, inflammatory cell accumulation, and collagen deposition. We investigated the effects of transgenic expression of human IGF-IA on gross lung histology, inflammatory cell numbers, and basal collagen matrix levels. Grossly, the transgenic mice were indistinguishable from their wild-type littermate controls. Baseline weights were not different (25.9 ± 1.5 g for wild-type animals vs. 25.5 ± 1.6 g for transgenic animals). Blinded review of lung histology by an expert lung pathologist revealed no appreciable differences between the transgenic mice and wild-type littermate controls, and both groups were characterized as normal mouse lung without atypical features or identifiable abnormalities (Fig. 3A). Quantification of the cellularity of the BALF revealed no significant differences between the human IGF-IA-expressing mice and wild-type littermate controls with regard to either total numbers of nucleated cells (P = 0.499) (Fig. 3B) or differential cell counts (Fig. 3C). In addition, basal levels of total lung collagen, as quantified by hydroxyproline levels, were not significantly different (P = 0.524) between the human IGF-IA-overexpressing mice and the wild-type littermates (Fig. 3D). Thus transgenic expression of human IGF-IA by alveolar type II epithelial cells had no demonstrable effect on basal measurements of gross lung pathology, bronchoalveolar lavage cellularity, or total lung collagen content.



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Fig. 3. Effect of transgenic expression of hIGF-IA on baseline lung histology, inflammatory cell accumulation, and collagen deposition. A: hematoxylin and eosin staining of sections of whole mouse lung preparations from IGF-IA transgene-expressing animals (IGF-ITG/–) and wild-type littermate controls. B: total numbers of nucleated cells in BALF. C: differential cell counts. PMN, polymorphonuclear neutrophils. D: basal levels of total lung collagen.

 
Effect of transgenic expression of human IGF-IA on bleomycin-induced pulmonary inflammation. We next investigated the effects of pulmonary overexpression of human IGF-IA on the development and progression of pulmonary inflammation in response to intratracheal instillation of bleomycin. Groups of human IGF-IA-expressing mice and wild-type littermate controls were instilled intratracheally with ~50 µl of bleomycin (4 U/kg) or saline vehicle. The mice were then subjected to bronchoalveolar lavage for analysis of BALF cellularity and IGF-I levels. All measurements were made at both 14 and 28 days after instillation. As can be seen in Fig. 4A, instillation of human IGF-IA-expressing mice with saline alone resulted in a significant decline in the level of human IGF-I detected in the BALF at 14 days (P < 0.0001). However, the level was restored to basal levels by 28 days. Bleomycin-instilled mice also showed a decline in BALF human IGF-I levels at 14 days compared with saline-instilled mice (P = 0.527). However, in contrast to saline instillation, the reduction in IGF-I levels was sustained at 28 days in mice that had been instilled with bleomycin (P = 0.01) (Fig. 4A).



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Fig. 4. Effect of transgenic expression of hIGF-IA on bleomycin (BLM)-induced pulmonary inflammation. A: hIGF-I levels in BALF; n = 72 total mice distributed among the 10 conditions with no condition containing fewer than n = 3. B: total nucleated cell counts in BALF; n = 98 total mice distributed among the 10 conditions with no condition containing fewer than n = 5. +, IGF-IA-expressing mice; –, wild-type littermate controls.

 
Figure 4B illustrates the changes in cellularity of the BALF in human IGF-IA-expressing mice and wild-type littermate controls in response to instillation of bleomycin or saline vehicle. Instillation of saline alone modestly increased the number of nucleated cells in BALF at 14 days but not at 28 days. In contrast, and as previously reported (23), intratracheal instillation of bleomycin into wild-type mice induced a significant increase in BALF cellularity at both 14 and 28 days (P < 0.0001). However, there was no significant difference (P = 0.869) in the BALF cellularity between the IGF-IA transgene-expressing mice and wild-type littermates (Fig. 4B). We also determined the differential cell counts from the BALF of all experimental conditions (Table 1). Marked increases in the numbers of foamy macrophages and lymphocytes were detected in response to instillation of bleomycin. However, no significant differences (all P values > 0.1) were detected in the number of these cells between human IGF-IA-expressing mice and wild-type littermate controls. Collectively, these data suggest that pulmonary inflammation in response to bleomycin is similar in human IGF-IA transgenic and wild-type mice.


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Table 1. Differential cell counts

 
Effect of transgenic expression of human IGF-IA on bleomycin-induced pulmonary fibrosis. Given that increased macrophage and alveolar epithelial cell expression of IGF-I has been detected in, and linked to, disease severity in IPF, we also investigated the effects of alveolar epithelial cell expression of human IGF-IA on the development of pulmonary fibrosis in the bleomycin model. As can be seen in Fig. 5A, instillation of bleomycin induced a significant increase (P < 0.0001) in total lung hydroxyproline levels in control wild-type littermates compared with either uninstilled mice or mice that were instilled with saline alone. Similar levels of hydroxyproline were detected at both 14 and 28 days. Intratracheal instillation of bleomycin into human IGF-IA transgenic mice also produced an increase in total lung hydroxyproline content. However, there was no significant difference in the hydroxyproline levels of lungs from human IGF-IA-expressing mice compared with wild-type littermate controls (P = 0.637). Similarly, histological sections of lungs from both the human IGF-IA-expressing mice and wild-type littermate controls showed characteristic bleomycin-induced lung injury with patchy interstitial thickening accompanied by increased numbers of fibroblasts, collagen deposition, and inflammation. Pathological review revealed no differences between the two groups (Fig. 5B).



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Fig. 5. Effect of transgenic expression of hIGF-IA on bleomycin-induced pulmonary inflammation. A: total lung hydroxyproline levels; n = 180 total mice distributed among the 10 conditions with no condition containing fewer than n = 12. B: hematoxylin and eosin staining of a lung section from an hIGF-IA transgene-expressing mouse and wild-type littermate control intratracheally instilled with bleomycin. +, IGF-IA-expressing mice; –, wild-type littermate controls.

 
Transgenic pulmonary expression of human IGF-IA promotes adenomatous hyperplasia. No abnormalities, including early tumor formation, were identified on pathological review of 8- to 12-wk-old mice. We therefore "aged" nine transgenic animals and eight wild-type littermate controls to greater than 1 yr of age (13–16 mo) and submitted the lungs for blinded, expert histopathological review. The histology of the older transgenic animals revealed significantly increased foci of adenomatous hyperplasia compared with wild-type littermate controls. Of the transgenic mice, nine of nine exhibited adenomatous hyperplasia compared with only four of eight of the wild-type littermate controls (P = 0.03). These foci were characterized by hyperplastic, hypertrophic airway epithelium lining small stretches of alveolar septae, and small airways (Fig. 6A). With regard to spontaneous pulmonary adenoma formation, six adenomas were identified among the nine transgenic animals for an incidence of 0.67 tumors/animal, whereas three were identified among the eight wild-type animals for an incidence of 0.375 tumors/animal (Fig. 6B). However, this difference did not achieve statistical significance (P = 0.454). The adenomas were characterized by nodular foci of hyperplastic and crowded epithelial cells (Fig. 6C). The nuclei of these cells were monotonous and without evidence of atypia or mitotic figures. There were focal clusters of macrophages within the air spaces of the larger adenomas. No appreciable differences in the histological type of tumor were noted between the transgenic mice and the wild-type littermate controls. Thus, in older mice, the overexpression of human IGF-IA appears to promote premalignant adenomatous hyperplastic lesions, precursors of adenomas.



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Fig. 6. A: transgenic expression of hIGF-IA promotes adenomatous hyperplasia characterized by hyperplastic, hypertrophic airway epithelium lining small stretches of alveolar septae and small airways. B: incidence of spontaneous lung adenoma formation in transgene-expressing animals and wild-type littermate controls. C: histological appearance of spontaneous pulmonary adenomas in older (>12 mo) mice.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Increased expression of IGF-I has been shown to be associated with prenatal lung development and alveolarization (21). IGF-I has also been strongly implicated in the pathophysiology of both IPF and non-small cell lung cancer (18, 35). To investigate the function of increased expression of IGF-I in alveolar air spaces, we created transgenic mice in which human IGF-IA was expressed under the control of the alveolar type II epithelial cell-specific SP-C promoter. We demonstrated that the mice had the expected genotype and characterized the effect of pulmonary expression of human IGF-IA on baseline pulmonary pathology. In addition, we investigated the effect of human IGF-I expression on the development of lung injury and fibrosis in response to intratracheal delivery of bleomycin. Pulmonary expression of human IGF-IA did not alter either baseline lung characteristics in young (8- to 12-wk-old) mice or the severity of bleomycin-induced lung injury or fibrosis. However, in older mice (>1 yr), the expression of human IGF-IA was found to augment the development of adenomatous hyperplasia, a precursor of adenoma and adenocarcinoma development.

In view of the extensive in vitro and in vivo data supporting a role for IGF-I in the development of pulmonary fibrosis, the lack of effect of pulmonary overexpression of human IGF-IA on bleomycin-induced pulmonary fibrosis was unexpected. We have considered four plausible explanations for these observations. First, IGF-I activity and availability are regulated by insulin-like growth factor binding proteins (IGFBP), which predominantly inhibit, but in the case of IGFBP-1, -3, and -5, may also enhance IGF-I activity (2). In particular, when cells are preincubated with IGFBP-3, IGF-I-dependent proliferation is enhanced, whereas the concurrent administration of IGFBP-3 with IGF-I results in diminished IGF-I effects (2). Moreover, IGFBP-3 and -5 have well-characterized IGF-I-independent effects (2). For example, IGF-I-independent, IGFBP-3-dependent induction of apoptosis is well described (2). Specifically as regards pulmonary fibrosis, IGFBP-2 and IGFBP-3 have been shown to be elevated in BALF in patients with IPF (1, 24). Therefore, bleomycin-induced changes in the levels of IGFBPs, especially IGFBP-2 and -3, could inhibit or mask the effect of IGF-I overexpression through decreased bioavailability of IGF-I or by IGF-I-independent mechanisms, as in the case of IGFBP-3.

Second, mature IGF-I is produced by proteolysis of either IGF-IA or IGF-IB. However, a recent study by Bloor et al. (4) showed that the expression of IGF-IA mRNA was decreased in bronchoalveolar cells from IPF subjects compared with normal controls, whereas the expression of IGF-IB transcripts was increased. Although this may initially seem inconsequential in terms of the production of mature IGF-I, studies by Siegfried et al. (32) have shown that the 77-residue E peptide produced by proteolysis of IGF-IB exhibits intrinsic mitogenic activity. In contrast, the E peptide from IGF-IA was inactive (32). It is therefore possible that the fibrogenic activity of IGF-I is specific to the IGF-IB splice variant and is not associated with IGF-IA.

Third, the SP-C promoter results in luminal secretion of transgenic IGF-I. Previous studies have shown that, in IPF, IGF-I is localized to CD68-positive alveolar and interstitial macrophages as well as to alveolar epithelial cells. However, associations between increased IGF-I expression and disease severity were only observed for IGF-I localization to interstitial macrophages (35), suggesting that IGF-I expression by interstitial macrophages may be functionally important in the development or progression of IPF. Likewise, studies in an asbestosis model in sheep have shown that transforming growth factor-{beta} expression is restricted to alveolar macrophages and epithelial cells, whereas IGF-I expression was also detected in interstitial cells (16). Similar compartment-specific expression patterns exist in the developing lung in which IGF-I, IGF-II, and IGF receptor levels vary considerably between mesenchymal, epithelial, and vascular tissues (29). Thus it is possible that air space delivery of human IGF-IA does not provide an appropriate localization for IGF-I to induce or increase pulmonary fibrosis.

Fourth, the fibrotic response is dependent on a large number of genes, cytokines, growth factors, and choreographed cellular responses, and thus it is possible that overexpressing a single gene product may not be sufficient to significantly alter the development of pulmonary fibrosis. Lastly, with regard to the lack of a significant difference in bleomycin-induced fibrosis between the two groups of mice, it is possible that the control animals have already achieved a peak effect with endogenous mouse IGF-I such that additional human IGF-I does not detectably alter the degree of observed fibrosis.

The decline seen in IGF-I levels in the BALF after bleomycin instillation was expected, as bleomycin has previously been shown to produce pronounced pulmonary inflammation and epithelial cell injury and apoptosis. However, the degree and extent of the declines in IGF-I levels after saline instillation were unexpected. Although we are not aware of any previous studies showing saline-induced effects on pulmonary inflammation, others have found that saline instillation induces a transient airway and/or air space inflammation lasting for several days (P. M. Henson, personal communication).

In contrast to its lack of effect on the development of pulmonary fibrosis, transgenic expression of human IGF-IA was found to increase premalignant adenomatous hyperplasia in old but not in young mice. Although there was a trend toward increased adenoma formation in the aged transgenic mice, this increase was not statistically significant. However, similar findings have been reported by DiGiovanni et al. (8) who examined the effects of overexpression of human IGF-I in mouse prostate epithelium. They found that overexpression of human IGF-I alone in the prostatic epithelium was sufficient to induce a step-wise progression of preneoplastic changes progressing from atypical hyperplasia to frank adenocarcinoma formation (8). Moreover, similar to the findings in our study, adenocarcinoma formation occurred exclusively in mice aged >6 mo and preferentially in mice aged >9 mo. Another recent study by Ng et al. (22) showed that exogenous administration of growth hormone and IGF-I to aging primates resulted in mammary gland hyperplasia, with four- to fivefold increases in epithelial cell proliferation and glandular size similar to that seen in the lung alveolar epithelial cells in the present study. Collectively, these findings implicate IGF-I in the pathogenesis of adenomatous hyperplasia and adenocarcinoma across organ systems.

Although the development of hyperplasia and adenomatous transformation is consistent with IGF-I's known mitogenic, trophic, and anti-apoptotic properties, the relative contributions of each of these pathways to these processes remain unclear. In the DiGiovanni study (8), no clear differences could be identified regarding proliferation or apoptosis between the transgenic animals and the wild-type controls. Still, variation among IGF-I and/or IGF-IR signaling pathways between different cell types has been previously documented, and extrapolation between different organ and cell types is difficult (28). Furthermore, with regard to our study in particular, whether the hyperplastic cells or adenomas observed in the transgenic animals specifically express IGF-IA and thereby stimulate their own growth and survival in an autocrine fashion or whether these cells are responding solely to IGF-IA secreted by neighboring epithelium in paracrine fashion raises interesting questions regarding the biology of IGF-I signaling. Finally, although IGF-I has also previously been shown to participate in aging (17), the synergistic interaction between aging and IGF-I that results in adenomatous transformation is intriguing, since previous studies in mesenchymal cells had shown decreased responsiveness to IGF-I stimulation with increasing age (33), Clearly, dissection of the mechanism(s) by which IGF-I promotes adenomatous transformation and the influence of age on IGF-I and/or IGF-IR signaling will need to be the focus of further study. Moreover, these collective findings should raise concerns regarding the therapeutic use of growth hormone and/or IGF-I in older individuals, such as the use of growth hormone for the treatment of cachexia in chronic obstructive pulmonary disease (5), until we have a better understanding of the effects of IGF-I on aging epithelium.

In summary, we report the development of transgenic mice expressing human IGF-IA under the influence of the SP-C promoter. Although no discernable effects could be identified on inflammation or fibrosis, a significant increase in premalignant epithelial cell hyperplasia was found in older mice.


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 GRANTS
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-68628 and HL-65326. S. K. Frankel was supported, in part, by K08 award HL-67848, a Glaxo-Wellcome Pulmonary Fellowship Grant, and an Andrew Goodman Fellowship in Pediatrics from National Jewish Medical and Research Center (Denver, CO). M. W. Wynes was supported by Institutional Training Grant T-32 AI-00048 from the National Institutes of Health.


    ACKNOWLEDGMENTS
 
The authors thank Benjamin L. Edelman and Linda K. Remigio for their excellent technical assistance and contributions to this work.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. W. H. Riches, Neustadt D-405, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206 (E-mail: richesd{at}njc.org)

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.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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