Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Conditional Expression of Fibroblast Growth Factor-7 in the Developing and Mature Lung

2000; Elsevier BV; Volume: 275; Issue: 16 Linguagem: Inglês

10.1074/jbc.275.16.11858

ISSN

1083-351X

Autores

Jay W. Tichelaar, Wei Lu, Jeffrey A. Whitsett,

Tópico(s)

Congenital Anomalies and Fetal Surgery

Resumo

Effects of fibroblast growth factor-7 (FGF-7) on lung morphogenesis, respiratory epithelial cell differentiation, and proliferation were assessed in transgenic mice in which the humanFGF-7 cDNA was controlled by a conditional promoter under the direction of regulatory elements from either the human surfactant protein-C (SP-C) or rat Clara cell secretory protein (ccsp) genes. Expression of FGF-7 was induced in respiratory epithelial cells of the fetal lung by administration of doxycycline to the dam. Prenatally, doxycycline inducedFGF-7 mRNA in respiratory epithelial cells in bothSp-c and Ccsp transgenic lines, increasing lung size and causing cystadenomatoid malformation. Postnatally, mice bearing both Ccsp-rtta and (Teto)7 -cmv-fgf-7 transgenes survived, and lung morphology was normal. Induction of FGF-7 expression by doxycycline in the Ccsp-rtta × (Teto)7 -cmv-fgf-7 mice caused marked epithelial cell proliferation, adenomatous hyperplasia, and pulmonary infiltration with mononuclear cells. Epithelial cell hyperplasia caused by FGF-7 was largely resolved after removal of doxycycline. Surfactant proteins, TTF-1, and aquaporin 5 expression were conditionally induced by doxycycline. The Sp-c-rttaand Ccsp-rtta activator mice provide models in which expression is conditionally controlled in respiratory epithelial cells in the developing and mature lung, altering lung morphogenesis, differentiation, and proliferation. Effects of fibroblast growth factor-7 (FGF-7) on lung morphogenesis, respiratory epithelial cell differentiation, and proliferation were assessed in transgenic mice in which the humanFGF-7 cDNA was controlled by a conditional promoter under the direction of regulatory elements from either the human surfactant protein-C (SP-C) or rat Clara cell secretory protein (ccsp) genes. Expression of FGF-7 was induced in respiratory epithelial cells of the fetal lung by administration of doxycycline to the dam. Prenatally, doxycycline inducedFGF-7 mRNA in respiratory epithelial cells in bothSp-c and Ccsp transgenic lines, increasing lung size and causing cystadenomatoid malformation. Postnatally, mice bearing both Ccsp-rtta and (Teto)7 -cmv-fgf-7 transgenes survived, and lung morphology was normal. Induction of FGF-7 expression by doxycycline in the Ccsp-rtta × (Teto)7 -cmv-fgf-7 mice caused marked epithelial cell proliferation, adenomatous hyperplasia, and pulmonary infiltration with mononuclear cells. Epithelial cell hyperplasia caused by FGF-7 was largely resolved after removal of doxycycline. Surfactant proteins, TTF-1, and aquaporin 5 expression were conditionally induced by doxycycline. The Sp-c-rttaand Ccsp-rtta activator mice provide models in which expression is conditionally controlled in respiratory epithelial cells in the developing and mature lung, altering lung morphogenesis, differentiation, and proliferation. fibroblast growth factor-7 5-bromodeoxyuridine surfactant protein-C Clara cell secretory protein FGF receptor kilobase pair polymerase chain reaction reverse transcriptase-PCR cytomegalovirus phosphate-buffered saline Fibroblast growth factor-7 (FGF-7)1 is a potent mitogen that enhances cell proliferation in various organs, including the skin, intestine, breast, liver, and lung. In the lung, FGF-7 and the related polypeptide FGF-10 are expressed in mesenchymal cells in distinct temporal and spatial patterns. Fgf-7 expression begins at embryonic day 14.5, being detected throughout the mesenchyme surrounding the developing lung tubules (1.Finch P.W. Cunha G.R. Rubin J.S. Wong J. Ron D. Dev. Dyn. 1995; 203: 223-240Crossref PubMed Scopus (264) Google Scholar, 2.Mason I.J. Fuller-Pace F. Smith R. Dickson C. Mech. Dev. 1994; 45: 15-30Crossref PubMed Scopus (262) Google Scholar). Fgf-10expression is initiated earlier, at the onset of lung organogenesis, and is restricted to the mesenchyme surrounding the distal tips of the branching tubules (3.Bellusci S. Grindley J. Emoto H. Itoh N. Hogan B.L.M. Development. 1997; 124: 4867-4878Crossref PubMed Google Scholar). FGF-7 and FGF-10 bind with high affinity to the same FGF receptor 2 (FGFR2-IIIb) isoform (4.Igarashi M. Finch P.W. Aaronson S.A. J. Biol. Chem. 1998; 273: 13230-13235Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar); however, FGF-10 also binds to FGFR1-IIIb (5.Luo Y. Lu W. Mohamedali K.A. Jang J.H. Jones R.B. Gabriel J.L. Kan M. McKeehan W.L. Biochemistry. 1998; 37: 16506-16515Crossref PubMed Scopus (51) Google Scholar). The expression of FGFR2 is restricted to the epithelial cells of the developing lung at the onset of lung organogenesis at embryonic day 9.5 and becomes increasingly restricted to peripheral respiratory epithelial cells as lung development progresses (6.Peters K.G. Werner S. Chen G. Williams L.T. Development. 1992; 114: 233-243Crossref PubMed Google Scholar, 7.Orr-Urtreger A. Givol D. Yayon A. Yarden Y. Lonai P. Development. 1991; 113: 1419-1434Crossref PubMed Google Scholar). The distinct patterns of expression ofFgf-7 and Fgf-10 mRNAs in lung mesenchyme and the complementary expression of Fgfr-2 in respiratory epithelial cells support a role for FGF signaling in lung morphogenesis. Experimental support for the role of FGF in epithelial/mesenchymal signaling during lung development has been obtained in vitroand in vivo. Ectopic FGF-7 caused cystadenomatoid malformations in the fetal lung, associated with increased chloride-dependent fluid secretion, cell proliferation, and disrupted branching morphogenesis in vivo and in vitro (8.Simonet W.S. DeRose M.L. Bucay N. Nguyen H.Q. Wert S.E. Zhou L. Ulich T.R. Thomason A. Danilenko D.M. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12461-12465Crossref PubMed Scopus (160) Google Scholar, 9.Zhou L. Graeff R.W. McCray Jr., P.B. Simonet W.S. Whitsett J.A. Am. J. Physiol. 1996; 271: L987-L994Crossref PubMed Google Scholar). Fgf-7 expression in the fetal lungin vivo was uniformly lethal by 16 days of gestation and was likely related to the massive cyst formation induced by the polypeptide. Conversely, genetic ablation of Fgf-7 did not cause pulmonary abnormalities (10.Guo L. Degenstein L. Fuchs E. Genes Dev. 1996; 10: 165-175Crossref PubMed Scopus (471) Google Scholar), indicating that this polypeptide was not required for normal lung morphogenesis, but rather may play a role in pulmonary homeostasis following injury. A primary role for FGF-10 in lung development was demonstrated by the finding that targeted disruption of Fgf-10 resulted in mice lacking lungs and limbs (11.Sekine K. Ohuchi H. Fujiwara M. Yamasaki M. Yoshizawa T. Sato T. Yagishita N. Matsui D. Koga Y. Itoh N. Kato S. Nat. Genet. 1999; 21: 138-141Crossref PubMed Scopus (991) Google Scholar, 12.Min H. Danilenko D.M. Scully S.A. Bolon B. Ring B.D. Tarpley J.E. DeRose M. Simonet W.S. Genes Dev. 1998; 12: 3156-3161Crossref PubMed Scopus (735) Google Scholar). In vitro, lung tubules migrate toward FGF-10-soaked beads, consistent with a chemotactic function for FGF-10 (13.Park W.Y. Miranda B. Lebeche D. Hashimoto G. Cardoso W.V. Dev. Biol. 1998; 201: 125-134Crossref PubMed Scopus (276) Google Scholar). Recently, creation of tetraploid fusion chimeras lacking embryonic expression of Fgfr-2 yielded mice with a lungless and limbless phenotype, similar to that seen inFgf-10 gene-targeted mice (14.Arman E. Haffner-Krausz R. Gorivodsky M. Lonai P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11895-11899Crossref PubMed Scopus (174) Google Scholar). FGF-7 is also a potent mitogen in the postnatal lung. Intratracheal instillation of FGF-7 caused transient, but marked, epithelial cell hyperplasia of both bronchiolar and alveolar epithelial cells in the lungs of rats and mice (15.Ulich T.R. Yi E.S. Longmuir K. Yin S. Biltz R. Morris C.F. Housley R.M. Pierce G.F. J. Clin. Invest. 1994; 93: 1298-1306Crossref PubMed Scopus (320) Google Scholar, 16.Zsengeller Z.K. Halbert C. Miller A.D. Wert S.E. Whitsett J.A. Bachurski C.J. Hum. Gene Ther. 1999; 10: 341-353Crossref PubMed Scopus (27) Google Scholar). Following FGF-7 treatment, cell proliferation peaks in 2–3 days, and lung structure and proliferative activity returns to normal 1–2 weeks after administration. Since FGF-7 has marked pro-mitotic activities in respiratory epithelial cells, its potential utility for prevention or treatment of lung injury has been explored in vivo. Pretreatment of the lung with FGF-7 protects animals from the effects of oxygen-, acid-, bleomycin-, or radiation-induced lung injury (17.Panos R.J. Bak P.M. Simonet W.S. Rubin J.S. Smith L.J. J. Clin. Invest. 1995; 96: 2026-2033Crossref PubMed Scopus (226) Google Scholar, 18.Yi E.S. Williams S.T. Lee H. Malicki D.M. Chin E.M. Yin S. Tarpley J. Ulich T.R. Am. J. Pathol. 1996; 149: 1963-1970PubMed Google Scholar, 19.Deterding R.R. Havill A.M. Yano T. Middleton S.C. Jacoby C.R. Shannon J.M. Simonet W.S. Mason R.J. Proc. Assoc. Am. Physicians. 1997; 109: 254-268PubMed Google Scholar, 20.Yano T. Deterding R.R. Simonet W.S. Shannon J.M. Mason R.J. Am. J. Respir. Cell Mol. Biol. 1996; 15: 433-442Crossref PubMed Scopus (144) Google Scholar). Additionally, FGF-7 has been shown to be increased in the lung in animal models of lung injury (21.Charafeddine L. D'Angio C.T. Richards J.L. Stripp B.R. Finkelstein J.N. Orlowski C.C. LoMonaco M.B. Paxhia A. Ryan R.M. Am. J. Physiol. 1999; 276: L105-L113PubMed Google Scholar), suggesting that FGF-7 has important functions in regulating the response of the lung to injury. Since Fgf-7 was uniformly fatal in utero when continuously expressed in the fetal lung, animal models that assess the chronic effects of FGF-7 on lung structure and function in vivo have not been developed. In the present work, transgenic mice were designed in which the expression of FGF-7 was placed under the control of a doxycycline-inducible gene control system (22.Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4268) Google Scholar), directed either by the 3.7-kb human SP-C or the 2.3-kb ratccsp gene promoters. By using this conditional system, fetal lethality observed in previous FGF-7 transgenic models was overcome. Induction of FGF-7 in utero altered branching morphogenesis and caused cystic dilation of the developing lung tubules. In the adult mouse lung, induction of FGF-7 altered gene expression caused widespread alveolar and bronchiolar hyperplasia that was largely reversible. Thertta construct was a gift of Dr. Herman Bujard (ZMBH, Heidelberg, Germany), and the 1-kb rtta coding sequence was placed under the control of the 3.7-kb human SP-C promoter (23.Glasser S.W. Korfhagen T.R. Wert S.E. Bruno M.D. McWilliams K.M. Vorbroker D.K. Whitsett J.A. Am. J. Physiol. 1991; 261: L349-L356PubMed Google Scholar) or the 2.3-kb rat ccsp promoter (24.Stripp B.R. Sawaya P.L. Luse D.S. Wikenheiser K.A. Wert S.E. Huffman J.A. Lattier D.L. Singh G. Katyal S.L. Whitsett J.A. J. Biol. Chem. 1992; 267: 14703-14712Abstract Full Text PDF PubMed Google Scholar) that selectively direct expression of transgenes in respiratory epithelial cells of the lung (Fig. 1). Polyadenylation sequences from SV40 or the human growth hormone gene were used to ensure transcript termination. Plasmid constructs were verified by sequencing and then microinjected into mouse oocytes using standard transgenic procedures. Transgenic mice were identified using PCR primers specific for each transgene as follows: 5′ primer inSP-C promoter, 5′-GAC ACA TAT AAG ACC CTG GTC A-3′; 5′ primer in ccsp promoter, 5′-ACT GCC CAT TGC CCA AAC AC-3′; 3′ primer in rtta coding sequence used for genotyping bothSp-c-rtta and Ccsp-rtta mice, 5′-AAA ATC TTG CCA GCT TTC CCC-3′. Primers used for identification of (Teto)7 -cmv-fgf-7 transgene are as follows: 5′ primer in CMV minimal promoter, 5′-GCC ATC CAC GCT GTT TTG-3′; 3′ primer in hFGF-7 coding region, 5′-CAT TTC CCC TCC GTT GTG-3′. Amplification of PCR product forSp-c-rtta and Ccsp-rtta was performed by denaturation at 94 °C for 5 min and then 30 cycles of amplification at 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, followed by a 7-min extension at 72 °C. Detection of (Teto)7 -cmv-fgf-7 was identical except the annealing temperature was 54 °C. Animals were housed under pathogen-free conditions in accordance with institutional guidelines. A 50× doxycycline HCl stock (25 mg/ml in 50% ethanol, Sigma) was freshly prepared prior to each administration of the drug. Oral doxycycline was administered in the drinking water at a final concentration of 0.5 mg/ml and 1% ethanol. Due to the light sensitivity of doxycycline, doxycycline water was replaced three times per week. Animals not receiving doxycycline were treated with vehicle only (1% ethanol final concentration). Lung tissue was homogenized in Tri-Zol reagent (Life Technologies, Inc.) and RNA isolated following manufacturer specifications. RT-PCR was used to detect transgene expression and distinguish between endogenous Fgf-7 and the human FGF-7 transgene. Reverse transcription was carried out on 1 μg of total lung RNA in the presence of 10 mmTris-HCl, pH 8.3, 50 mm KCl, 2.5 mmMgCl2, 0.5 mm each dNTP, and 25 ng/μl oligo(dT) at 42 °C for 1 h. PCR primers for β-actin are as follows: β-actin primer 1, 5′-GTG GGC CGC TCT AGG CAC CAA-3′; β-actin primer 2, 5′-CTC TTT GAT GTC ACG CAG GAT TTC-3′; for humanFGF-7: hFGF-7 primer 1, 5′-ATA TCA TGG AAA TCA GGA CA-3′; hFGF-7 primer 2, 5′-CAT TTC CCC TCC GTT GTG-3′. PCR conditions for hFGF-7 were 94 °C denaturation for 5 min, followed by 25 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, followed by a 7-min extension at 72 °C. PCR conditions for β-actin were similar, but the annealing temperature was 59 °C. All reactions were run in duplicate with a negative control reaction lacking reverse transcriptase enzyme. No amplification was seen in reactions lacking reverse transcriptase (data not shown). Lungs were inflation fixed using 4% paraformaldehyde at 25 cm of pressure and then allowed to fix overnight at 4 °C, washed with phosphate-buffered saline (PBS), dehydrated through a graded series of ethanols, and processed for paraffin embedding. 5-μm sections were loaded onto polylysine slides for immunostaining. Antibodies and procedures for immunostaining of TTF-1 and pro-SP-C have been previously described (25.Zhou L. Lim L. Costa R.H. Whitsett J.A. J. Histochem. Cytochem. 1996; 44: 1183-1193Crossref PubMed Scopus (248) Google Scholar). The AQP5 polyclonal antibody, a gift from Dr. Anil Menon (University of Cincinnati), was used at a 1:1000 dilution and has been previously characterized (26.Krane C.M. Towne J.E. Menon A.G. Mamm. Genome. 1999; 10: 498-505Crossref PubMed Scopus (55) Google Scholar). For immunohistochemical detection of BrdUrd, animals were injected with 0.1 ml of BrdUrd labeling reagent (Zymed Laboratories Inc.) per 100 × g of body weight 2 h prior to sacrifice. BrdUrd incorporated into DNA was detected using anti-BrdUrd monoclonal antibody and a BrdUrd staining kit (Zymed Laboratories Inc.). Volume of inflated lungs was determined after overnight fixation and an initial PBS wash. Inflated lungs were dissected from surrounding tissue and weighed by immersion in PBS. Paraffin-embedded lung tissue was cut at 5 μm and loaded onto silanized slides. Sense and antisense riboprobes were generated from a murine Fgf-7 cDNA orrtta cDNA cloned into pGEM3z and transcribed in vitro with a Riboprobe transcription kit (Promega). Conditions and solutions for hybridization are essentially as described (27.Wert S.E. Glasser S.W. Korfhagen T.R. Whitsett J.A. Dev. Biol. 1993; 156: 426-443Crossref PubMed Scopus (275) Google Scholar). For the FGF-7 sense and antisense probes, hybridization was carried out overnight at 42 °C and washed under low stringency conditions. For the rtta sense and antisense probes, hybridization was carried out overnight at 55 °C and washed under high stringency conditions. Slides were dipped in Kodak NTB2 emulsion and exposed for 3–7 days. Slides were developed with Kodak D19 developer following the manufacturer's protocol. Transgenic mice were produced bearing the reverse tetracycline-responsive transactivator (rtta) fusion protein under control of the 3.7-kb human SP-C gene promoter or the 2.3-kb rat ccsp gene promoter (Fig.1), hereafter referred to asSp-c or Ccsp “activator” mice. The resulting founders were bred to establish permanent lines, screening for expression of the rtta transgene by RT-PCR and Northern blotting. Transgene expression was detected in only 1 of 15Sp-c-rtta and 1 of 7 Ccsp-rtta founders (data not shown). Separate transgenic mouse lines were generated by microinjection of a (Teto)7 -cmv-fgf-7transgene (Fig. 1 A), and two separate mouse lines bearing the target transgene were established. The (Teto)7 -cmv-fgf-7 transgene consists of seven copies of the tet operator DNA binding sequence linked to a minimal CMV promoter (22.Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4268) Google Scholar), the human FGF-7 cDNA, and SV40 polyadenylation signals. Transgenic mice bearing either theSp-c-rtta, Ccsp-rtta, or (Teto)7 -cmv-fgf-7 transgenes had no lung or other tissue pathologies and survived normally in the vivarium. To obtain transgenic mice in which FGF-7 expression was regulated by administration of doxycycline, Sp-c orCcsp activator mice were bred to (Teto)7 -cmv-fgf-7 target mice producing bitransgenic progeny (Fig. 1 B). To determine the effects of inducing FGF-7 expression in utero utilizing the Ccsp-rtta activator mice, double transgenic Ccsp-rtta × (Teto)7 -cmv-fgf-7 mice were generated. Fetal pups were obtained at embryonic day 17 after 7 days of doxycycline administration to the dam. Histological examination of the lungs from doxycycline treated Ccsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic pups revealed marked cystadenomatoid malformation similar to, but less severe than, that induced by FGF-7 previously (Fig.2 B) (8.Simonet W.S. DeRose M.L. Bucay N. Nguyen H.Q. Wert S.E. Zhou L. Ulich T.R. Thomason A. Danilenko D.M. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12461-12465Crossref PubMed Scopus (160) Google Scholar). Lung histology in littermates bearing only the (Teto)7 -cmv-fgf-7 transgene was normal (Fig. 2 A). Cystadenomatoid malformations were detected in the lungs ofSp-c-rtta × (Teto)7 -cmv-fgf-7 mice in the absence of doxycycline (Fig. 2 D). Consistent with this finding, human FGF-7 mRNA was detected in the lungs of these double transgenic mice in the absence of doxycycline but was increased 24 h after administration of doxycycline to the dam's drinking water (Fig. 2, G and H). TransgenicFGF-7 mRNA was not detected in single transgenic (Teto)7 -cmv-fgf-7 (Fig.2 F) or Sp-c-rtta (not shown) transgenic mice in the presence or absence of doxycycline. Without doxycycline, double transgenic Sp-c-rtta × (Teto)7 -cmv-fgf-7 pups survived until birth but died in the immediate postnatal period, likely relating to the abnormalities in lung structure and function. Because of the expression of FGF-7 mRNA in Sp-c-rtta × (Teto)7 -cmv-fgf-7 double transgenic mice in the absence of doxycycline, Ccsp-rtta activator mice were used for subsequent studies. In the absence of doxycycline, double transgenic Ccsp-rtta × (Teto)7 -cmv-fgf-7 mice survived postnatally, and no abnormalities in lung morphology were observed (Fig. 3 B). Administration of doxycycline to the drinking water of Ccsp-rtta × (Teto)7 -cmv-fgf-7 mice caused marked respiratory epithelial hyperplasia in the lungs of adult mice (Fig.3 C). No pulmonary abnormalities were observed inCcsp-rtta or (Teto)7 -cmv-fgf-7 single transgenic mice treated with doxycycline (Fig. 3 A) or in bitransgenic mice that did not receive doxycycline (Fig. 3 B). Hyperplasia of the alveolar epithelium was first observed after 4 days of treatment with doxycycline (not shown) and was most pronounced near the pleural surface and surrounding conducting airways. Seven days after treatment with doxycycline, epithelial cell hyperplasia became more widespread. The bronchial and bronchiolar epithelium was thickened and was often lined by a pseudostratified rather than columnar epithelium (Fig.3 C). Epithelial cell hyperplasia was increasingly widespread 14 or 21 days after exposure to doxycycline (Fig. 3, E andG). The lungs of Ccsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice contained increased numbers of alveolar macrophages that increased with continued doxycycline exposure (Fig. 3 D). Additionally, the interstitial space between conducting airways, blood vessels, and lung parenchyma was typically widened, becoming more pronounced with increasing duration of doxycycline administration (Fig. 3, Cand E). There was no evidence of fibrosis or collagen deposition as assessed by trichrome staining (data not shown). AdultCcsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice treated with doxycycline for 21 days had dramatically enlarged lungs, although their body weights were less than untreated bitransgenic or doxycyclin-treated (Teto)7 -cmv-fgf-7single transgenic littermates (Fig. 4). Continued treatment of bitransgenic mice with doxycycline caused respiratory distress and was generally lethal by 21 days.Figure 4Lungs from Ccsp-rtta × (Teto)7 -cmv-fgf-7bitransgenic mice. Lungs were inflation fixed at 25 cm of pressure from (Teto)7 -cmv-fgf-7single transgenic mice treated with doxycycline for 21 days (tetO-FGF-7, 21 d Dox), untreatedCcsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice (Dbl. Tg., − Dox), or Ccsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice treated with doxycycline for 21 days. After overnight fixation PBS displacement was used to determine lung volume (graph).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In situ hybridization with a mouse Fgf-7 riboprobe was used to detect FGF-7 mRNA in lung tissues of transgenic mice. In the absence of doxycycline, FGF-7 mRNA was undetectable in the lungs of Ccsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice and in single transgenic littermates treated with doxycycline (Fig.5, A and B). Treatment of Ccsp-rtta × (Teto)7 -cmv-fgf-7 mice for 7 days with doxycycline increased FGF-7 mRNA in conducting airway and alveolar epithelial cells (Fig. 5 C). In situ hybridization was used to identify cells expressing the rtta transgene under the control of the ratccsp promoter. In Ccsp-rtta transgenic mice, rtTA mRNA was readily detected in bronchial and type II epithelial cells (Fig. 5 E) in a pattern nearly identical to that seen inCcsp-rtta × (Teto)7 -cmv-fgf-7 double transgenic mice in the absence of doxycycline (Fig. 5 F). Treatment of double transgenic mice for 7 days with doxycycline increasedRtta mRNA in respiratory epithelial cells in a pattern similar to that of the Fgf-7 transgene (Fig. 5 G). Thus, treatment of Ccsp-rtta × (Teto)7 -cmv-fgf-7 double transgenic mice with doxycycline induced both FGF-7 and RttamRNAs. Incorporation of BrdUrd into DNA was used to estimate cell proliferation in the transgenic mice. Although no changes in BrdUrd uptake were detected in the lungs of Ccsp-rtta× (Teto)7 -cmv-fgf-7 mice 24 h after doxycycline (not shown), increased BrdUrd labeling of epithelial cells was noted after 4 days of doxycycline (Fig.6 B), the number of BrdUrd-labeled cells increased further after 7 days (Fig.6 C). BrdUrd staining was detected in respiratory epithelial cells in the lung periphery, although BrdUrd-labeled cells were occasionally detected in bronchial and bronchiolar epithelial cells. Administration of doxycycline to double transgenicCcsp-rtta × (Teto)7 -cmv-fgf-7 mice increased the intensity of staining for TTF-1 (Fig. 7,C and D) and pro-SP-C (Fig. 7, A andB), the latter a specific marker for type II epithelial cells in the lung periphery. Both pro-SP-C and TTF-1 were readily detectable in the lungs of bitransgenic mice treated with doxycycline at dilutions of antibody at which little or no signal was detected in double transgenic littermates that did not receive doxycycline (Fig. 7,C and D). Respiratory epithelial markers SP-B, pro-SP-B, and HNF-3β were also increased in doxycycline-treated bitransgenic animals compared with untreated bitransgenic littermates (data not shown). Additionally, immunostaining for the CCSP protein, a Clara cell marker, was maintained in the hyperplastic bronchial and bronchiolar epithelium and was not detected in regions of alveolar hyperplasia (data not shown). Aquaporin 5 (AQP5) and proSP-C staining was increased 7 days after treatment with doxycycline (Fig. 7,B and F), AQP5 being detected in cuboidal and squamous epithelial cells in the lung periphery as well as bronchiolar epithelial cells. The hyperplastic epithelial cells stained heavily for AQP5 on apical membranes.Figure 7Immunohistochemical staining for proSP-C, TTF-1, and AQP5. Staining for pro-SP-C (A andB), TTF-1 (C and D) and AQP5 (E and F) in adult lung tissue ofCcsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice without doxycycline treatment (A, C, and E) or following 7 days of doxycycline treatment (B, D, andF). Staining for pro-SP-C, a marker for type II alveolar epithelial cells, and for TTF-1 was increased inCcsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice treated with doxycycline (Dox) for 7 days compared with untreated littermates. Staining for AQP5, normally found in squamous type I alveolar epithelial cells, was abundant in cuboidal type II alveolar epithelial cells and in bronchial epithelial cells following treatment of Ccsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice with doxycycline for 7 days. b, bronchi. Figure is representative of at least two mice from each genotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ccsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice were exposed to doxycycline in the drinking water for 1–21 days. Transgene expression was assessed using primers that amplified the human FGF-7 cDNA but not the mouse Fgf-7cDNA. In the absence of doxycycline, human FGF-7mRNA was undetectable (Fig.8 A). In contrast, humanFGF-7 mRNA was readily detected after 24 h of doxycycline and increased during the chronic administration of doxycycline. Mice that had been treated with doxycycline for 7 days were provided water without doxycycline and studied for 1–7 days thereafter. Human FGF-7 mRNA was decreased 1 day after removal of doxycycline and thereafter (Fig. 8 B). Lung histology was assessed in Ccsp-rtta × (Teto)7 -cmv-fgf-7 double transgenic mice after cessation of doxycycline treatment. Respiratory epithelial hyperplasia was substantially reduced 7 days after cessation of doxycycline (not shown) and was nearly resolved 14 days after cessation of doxycycline (Fig. 3, F and H), with nearly complete restoration of normal lung parenchymal morphology. We have established a system for conditional expression ofFGF-7 in the lungs of transgenic mice, overcoming the prenatal lethality associated with expression of FGF-7 in the developing lung in vivo. Treatment of fetal or adult mice with doxycycline caused a rapid induction of FGF-7mRNA, resulting in cystadenomatoid malformation in fetal lung and epithelial hyperplasia in adult lung. In the adult lung, FGF-7 increased BrdUrd uptake and staining for pro-SP-C and TTF-1, consistent with proliferation and differentiation of type II epithelial cells. The effects of FGF-7 on cellular hyperplasia and targeted gene expression were largely reversed after removal of doxycycline. TheCcsp-rtta and Sp-c-rtta activator mice will likely be useful in generating mouse models in which diverse biologically active molecules are selectively expressed under control of exogenous doxycycline in developing or mature lung. FGF-7 caused respiratory epithelial hyperplasia and increased staining for a number of type II epithelial markers including TTF-1, HNF-3β, proSP-C, and SP-B. Epithelial hyperplasia was first seen in Ccsp-rtta × (Teto)7 -cmv-fgf-7 bitransgenic mice 4 days after doxycycline treatment and persisted for up to 3 weeks. In contrast, FGF-7 caused epithelial cell proliferation and hyperplasia 3 days after intratracheal administration that resolved within 7 days (15.Ulich T.R. Yi E.S. Longmuir K. Yin S. Biltz R. Morris C.F. Housley R.M. Pierce G.F. J. Clin. Invest. 1994; 93: 1298-1306Crossref PubMed Scopus (320) Google Scholar). Whereas the effects of intratracheal FGF-7 were noted primarily in the alveolar region, hyperplasia was also noted in the conducting airways after doxycycline exposure in our model, demonstrating that conducting airway epithelial cells are also responsive to the growth factor. Pleural thickening, associated with increased BrdUrd uptake, was also observed after chronic expression of FGF-7. The present observations are consistent with the finding that antibodies to FGF-7 blocked stimulatory effects of pleural lavage fluid from asbestos-treated rats on pleural mesothelial cells (28.Adamson I. Prieditis H. Young L. Am. J. Respir. Cell Mol. Biol. 1997; 16: 650-656Crossref PubMed Scopus (17) Google Scholar). The interstitial space surrounding conducting airways and blood vessels was enlarged in Ccsp-rtta × (Teto)7 -cmv-fgf-7 mice treated with doxycycline although the interstitium was not fibrotic. The lack of cells or extracellular matrix accumulation suggests that the increase in interstitial space may result from fluid accumulation. This possibility is supported by a dramatic increase and redistribution of AQP5 from its normal expression in type I alveolar epithelial cells to the hyperplastic alveolar epithelial cells found inCcsp-rtta × (Teto)7 -cmv-fgf-7 double transgenic mice. Previous studies demonstrated that FGF-7 regulates fluid balance in the fetal lung (8.Simonet W.S. DeRose M.L. Bucay N. Nguyen H.Q. Wert S.E. Zhou L. Ulich T.R. Thomason A. Danilenko D.M. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12461-12465Crossref PubMed Scopus (160) Google Scholar, 9.Zhou L. Graeff R.W. McCray Jr., P.B. Simonet W.S. Whitsett J.A. Am. J. Physiol. 1996; 271: L987-L994Crossref PubMed Google Scholar, 29.Graeff R.W. Wang G. McCray Jr., P.B. Pediatr. Res. 1999; 46: 523-529Crossref PubMed Scopus (23) Google Scholar), and the present findings suggest that FGF-7 regulates AQP5 expression, an epithelial water channel. The increased expression of AQP5 in vivo is in contrast to a previous report demonstrating that FGF-7 inhibited Aqp5expression during in vitro culture of alveolar type II cells (30.Borok Z. Lubman R.L. Danto S.I. Zhang X.L. Zabski S.M. King L.S. Lee D.M. Agre P. Crandall E.D. Am. J. Respir. Cell Mol. Biol. 1998; 18: 554-561Crossref PubMed Scopus (164) Google Scholar). The distinct effects of FGF-7 on Aqp5 expression in the two systems may represent different responses of alveolar epithelial cells in vitro versus in vivo. Pulmonary edema is common in lung injury, and increased FGF-7 may be involved in resolution of edema. Consistent with this hypothesis, it is known that FGF-7 decreases pulmonary edema (31.Yi E.S. Salgado M. Williams S. Kim S.J. Masliah E. Yin S. Ulich T.R. Inflammation. 1998; 22: 315-325Crossref PubMed Scopus (62) Google Scholar), probably by increasing alveolar liquid clearance (32.Wang Y. Folkesson H. Jayr C. Ware L. Matthay M. J. Appl. Physiol. 1999; 87: 1852-1860Crossref PubMed Scopus (92) Google Scholar). In vitro, or following intratracheal doses, FGF-7 also regulates other mediators of fluid balance such as the epithelial sodium channel, the sodium/potassium-ATPase (Na+/K+-ATPase), and the epithelial chloride channel ClC-2 (33.Guery B.P. Mason C.M. Dobard E.P. Beaucaire G. Summer W.R. Nelson S. Am. J. Respir. Crit. Care Med. 1997; 155: 1777-1784Crossref PubMed Scopus (80) Google Scholar, 34.Borok Z. Danto S.I. Dimen L.L. Zhang X.L. Lubman R.L. Am. J. Physiol. 1998; 274: L149-L158Crossref PubMed Google Scholar, 35.Blaisdell C.J. Pellettieri J.P. Loughlin C.E. Chu S. Zeitlin P.L. Am. J. Respir. Cell Mol. Biol. 1999; 20: 842-847Crossref PubMed Scopus (22) Google Scholar, 36.Borok Z. Mihyu S. Fernandes V.F. Zhang X.L. Kim K.J. Lubman R.L. Am. J. Physiol. 1999; 276: C1352-C1360Crossref PubMed Google Scholar). FGF-7 caused a marked increase in the number and BrdUrd labeling of alveolar macrophages. It is unclear whether this represents a direct effect of FGF-7 on macrophage recruitment or proliferation of alveolar precursors or is a response to the epithelial cell hyperplasia and remodeling of lung parenchyma. Chronic stimulation of FGF-7 in the lung caused severe hyperplasia that resembled a pre-cancerous human lesion, termed alveolar adenomatous hyperplasia (37.Colby T.V. Brambilla C. Brambilla E. Lung Tumors: Fundamental Biology and Clinical Management. 124. Marcel Dekker, Inc., New York1999: 61-87Google Scholar). Withdrawal of doxycycline caused nearly complete remodeling of the lung, although mild epithelial cell hyperplasia was observed 14 days after cessation of doxycycline. Resolution of FGF-7-induced hyperplasia is thought to be mediated by apoptosis of type II cells and differentiation of type II cells into type I cells (38.Fehrenbach H. Kasper M. Tschernig T. Pan T. Schuh D. Shannon J.M. Muller M. Mason R.J. Eur. Respir. J. 1999; 14: 534-544Crossref PubMed Scopus (91) Google Scholar). After removal of doxycycline, the normal pattern of immunostaining of APQ5 in squamous epithelial cells was restored, consistent with differentiation of type II cells into type I cells. The reversible hyperplasia seen in our model agrees with recent transgenic models of neoplasia (39.Xie W. Chow L.T. Paterson A.J. Chin E. Kudlow J.E. Oncogene. 1999; 18: 3593-3607Crossref PubMed Scopus (123) Google Scholar, 40.Chin L. Tam A. Pomerantz J. Wong M. Holash J. Bardeesy N. Shen Q. O'Hagan R. Pantginis J. Zhou H. Horner J.W., II Cordon-Cardo C. Yancopoulos G.D. DePinho R.A. Nature. 1999; 400: 468-472Crossref PubMed Scopus (760) Google Scholar, 41.Felsher D.W. Bishop J.M. Mol. Cell. 1999; 4: 199-207Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar) and suggests that inappropriate expression of FGF-7 was not oncogenic. TheRtta mRNA was detected in type II alveolar epithelial cells as well as in conducting airway epithelial cells inCcsp-rtta single transgenic mice. The presence ofRtta mRNA in type II cells was somewhat surprising since the ccsp promoter typically directs transgene expression to non-ciliated epithelial cells in the conducting airway. We have previously observed type II cell expression using the ratccsp promoter in transgenic mice, perhaps reflecting species differences or positional effects that influence the sites of expression. 2J. W. Tichelaar, S. Wert, and J. A. Whitsett, unpublished observations. FGF-7 caused proliferation of alveolar epithelial cells expressing the (Teto)7 -cmv-fgf-7 transgene and also increased staining for TTF-1 within these cells. It is therefore possible that FGF-7 increased Ttf-1 expression that, in turn, may have influenced the level of activity of the ratccsp promoter per se (42.Zhang L. Whitsett J.A. Stripp B.R. Biochim. Biophys. Acta. 1997; 1350: 359-367Crossref PubMed Scopus (127) Google Scholar), increasing levels ofRtta that may further induce the activity of the (Teto)7 -cmv-fgf-7 transgene. Conditional expression of FGF-7 in the fetal lung caused cystadenomatoid malformation in vivo, consistent with previous in vitro and in vivo findings (8.Simonet W.S. DeRose M.L. Bucay N. Nguyen H.Q. Wert S.E. Zhou L. Ulich T.R. Thomason A. Danilenko D.M. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12461-12465Crossref PubMed Scopus (160) Google Scholar, 9.Zhou L. Graeff R.W. McCray Jr., P.B. Simonet W.S. Whitsett J.A. Am. J. Physiol. 1996; 271: L987-L994Crossref PubMed Google Scholar). The severity of malformation induced by Ccsp-rtta as compared with Sp-c-rtta may be related to the timing and level of expression, the SP-C promoter being active earlier in gestation (27.Wert S.E. Glasser S.W. Korfhagen T.R. Whitsett J.A. Dev. Biol. 1993; 156: 426-443Crossref PubMed Scopus (275) Google Scholar). Although lung abnormalities were not observed inCcsp-rtta-activated mice in the absence of doxycycline, cystic abnormalities were seen in the Sp-c-rtta-activated mice in the absence of doxycycline. The ability of low levels of FGF-7 to induce a cystic phenotype is consistent with the potent effect of FGF-7 on respiratory epithelial cells (43.Deterding R.R. Jacoby C.R. Shannon J.M. Am. J. Physiol. 1996; 271: L495-L505Crossref PubMed Google Scholar). FGF-7 expression was induced in the fetal lung of Sp-c-rtta × (Teto)7 -cmv-fgf-7 bitransgenic pups by the addition of doxycycline to the drinking water of the dam, indicating that the system remains inducible and of potential utility for study of lung morphogenesis and function. FGF-7 and FGF-10 both bind to the FGFR2(IIIb) receptor found on respiratory epithelial cells, predicting similar phenotypes following induction of these growth factors in the lung. However, effects of FGF-7 on lung morphogenesis are distinct from those caused by FGF-10, 3J. C. Clark, J. W. Tichelaar, N. Itoh, A.-K. T. Perl, S. E. Wert, M. T. Stahlman, and J. A. Whitsett, unpublished observations. suggesting that signaling by these closely related FGF polypeptides utilizes distinct pathways or modifiers. Recently it was shown that FGF-10, but not FGF-7, binds to FGFR1(IIIb) (5.Luo Y. Lu W. Mohamedali K.A. Jang J.H. Jones R.B. Gabriel J.L. Kan M. McKeehan W.L. Biochemistry. 1998; 37: 16506-16515Crossref PubMed Scopus (51) Google Scholar), a receptor present in pulmonary mesenchyme (1.Finch P.W. Cunha G.R. Rubin J.S. Wong J. Ron D. Dev. Dyn. 1995; 203: 223-240Crossref PubMed Scopus (264) Google Scholar, 7.Orr-Urtreger A. Givol D. Yayon A. Yarden Y. Lonai P. Development. 1991; 113: 1419-1434Crossref PubMed Google Scholar). Whether the distinct effects of FGF-7 and FGF-10 on lung morphogenesis are influenced by FGF-10-mediated pathways in mesenchyme remains to be elucidated. Heparan sulfate proteoglycans influence signaling through FGFRs (44.Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and the patterns of heparan sulfate expression and the extent of their glycosylation vary developmentally (45.Brauker J. Trautman M. Bernfield M. Dev. Biol. 1991; 147: 285-292Crossref PubMed Scopus (43) Google Scholar). Thus, the differences in phenotype inFGF-7 and FGF-10 expressing mice may reflect differences in receptor activation by each ligand related to developmental changes in heparan sulfate proteoglycans or other modifiers of FGF-signaling pathways in target cells. Generation of Ccsp-rtta × (Teto)7 -cmv-fgf-7 double transgenic mice provides a tightly regulated system for studying the effects of FGF-7 in the respiratory epithelium. The utility of the Ccspsystem is evidenced by the finding that the human FGF-7transgene was not detected in unstimulated Ccsp-rtta × (Teto)7 -cmv-fgf-7 mouse lung and is further supported by studies using (tetO)7-CMV-luciferase mice to assay levels of Ccsp-rtta regulated transgene induction. 4A.-K. T. Perl, J. W. Tichelaar, and J. A. Whitsett, manuscript in preparation. Transgene expression was not detected in tissues other than lung, and cell proliferation was not increased in the livers of Ccsp-rtta × (Teto)7 -cmv-fgf-7 mice treated with doxycycline (data not shown), indicating that the activity of FGF-7 was restricted to the lung. The present findings support previous studies demonstrating doxycycline-regulated expression of IL-11 in mice co-injected with both Ccsp-rtta and (tetO)7-CMV-IL-11 transgenes (46.Ray P. Tang W. Wang P. Homer R. Kuhn III, C. Flavell R.A. Elias J.A. J. Clin. Invest. 1997; 100: 2501-2511Crossref PubMed Scopus (129) Google Scholar). In those studies, inducible expression of IL-11 and pulmonary abnormalities were observed following doxycycline treatment, although IL-11 was detected in the absence of doxycycline. SinceSp-c-rtta and Ccsp-rtta were generated as independent activator lines, these mice can be bred to various “target” mice conferring conditional expression of exogenous genes in the lung. We thank Drs. P. Ray and J. Elias for helpful discussion and Dr. H. Bujard for the availability of the rtTA and (tetO)7CMV plasmids.

Referência(s)