Excess FGF-7 in Corneal Epithelium Causes Corneal Intraepithelial Neoplasia in Young Mice and Epithelium Hyperplasia in Adult Mice
2008; Elsevier BV; Volume: 172; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2008.070897
ISSN1525-2191
AutoresTai-ichiro Chikama, Chia‐Yang Liu, Johanna T. A. Meij, Yasuhito Hayashi, I‐Jong Wang, Liu Yang, Teruo Nishida, Winston W.‐Y. Kao,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoWe hypothesized that human ocular surface squamous neoplasia (OSSN) may result from the continuous growth stimulation of corneal epithelial progenitor cells. In the present study, we analyzed the effects of excess fibroblast growth factor-7 (FGF-7) on both the proliferation and differentiation of corneal epithelium in a novel Krt12-rtTA/tet-O-FGF-7 double transgenic mouse model in which cornea-specific FGF-7 overexpression is achieved by doxycycline (Dox) treatment. When such adult mice were exposed to Dox, they exhibited epithelial hyperplasia with increases in phospho-extracellular signal-regulated kinase 1/2-, nuclear β-catenin-, and 5-bromo-2′-deoxyuridine-labeled cells and altered keratin (K) 14 (K14) expression pattern, a normal K12 expression pattern, and the normal absence of K10. Hyperplasia of the adult cornea was fully reversible 2 weeks after the removal of Dox from chow. In contrast, double transgenic embryos that were exposed to Dox from embryonic day 0.5 to postnatal day 21 developed papillomatous tumors in the cornea, resembling human OSSN, and ectopic gland-like structures in the limbus, accompanied by the down-regulation of K12 and the up-regulation of K14, Pax6, and p63. These epithelial anomalies observed in young experimental mice were not fully resolved after the termination of Dox induction. Taken together, Krt12-rtTA/tet-O-FGF-7 mice may be a suitable animal model for the study of the molecular and cellular mechanisms of human OSSN. We hypothesized that human ocular surface squamous neoplasia (OSSN) may result from the continuous growth stimulation of corneal epithelial progenitor cells. In the present study, we analyzed the effects of excess fibroblast growth factor-7 (FGF-7) on both the proliferation and differentiation of corneal epithelium in a novel Krt12-rtTA/tet-O-FGF-7 double transgenic mouse model in which cornea-specific FGF-7 overexpression is achieved by doxycycline (Dox) treatment. When such adult mice were exposed to Dox, they exhibited epithelial hyperplasia with increases in phospho-extracellular signal-regulated kinase 1/2-, nuclear β-catenin-, and 5-bromo-2′-deoxyuridine-labeled cells and altered keratin (K) 14 (K14) expression pattern, a normal K12 expression pattern, and the normal absence of K10. Hyperplasia of the adult cornea was fully reversible 2 weeks after the removal of Dox from chow. In contrast, double transgenic embryos that were exposed to Dox from embryonic day 0.5 to postnatal day 21 developed papillomatous tumors in the cornea, resembling human OSSN, and ectopic gland-like structures in the limbus, accompanied by the down-regulation of K12 and the up-regulation of K14, Pax6, and p63. These epithelial anomalies observed in young experimental mice were not fully resolved after the termination of Dox induction. Taken together, Krt12-rtTA/tet-O-FGF-7 mice may be a suitable animal model for the study of the molecular and cellular mechanisms of human OSSN. Ocular surface squamous neoplasia (OSSN) is rare, but it is the most common ocular surface precancerous and cancerous lesion previously known by various names, such as conjunctival intraepithelial neoplasia, corneal intraepithelial neoplasia, or both together.1Grossniklaus HE Green WR Lukenback M Chan CC Conjunctival lesions in adults: a clinical and histopathologic review.Cornea. 1987; 6: 78-116Crossref PubMed Scopus (281) Google Scholar Clinically, OSSN manifests in different grades ranging from simple dysplasia to squamous cell carcinoma.1Grossniklaus HE Green WR Lukenback M Chan CC Conjunctival lesions in adults: a clinical and histopathologic review.Cornea. 1987; 6: 78-116Crossref PubMed Scopus (281) Google Scholar Because of the high incidence of OSSN in the limbal area, where the corneal epithelial stem cells are located, the limbal transition zone/stem cell theory has been proposed for the development of corneal intraepithelial neoplasia by Lee and Hirst.2Lee GA Hirst LW Ocular surface squamous neoplasia.Surv Ophthalmol. 1995; 39: 429-450Abstract Full Text PDF PubMed Scopus (465) Google Scholar Tseng et al3Tseng SCG Concept and application of limbal stem cells.Eye. 1989; 3: 141-157Crossref PubMed Scopus (526) Google Scholar have suggested that the slow cycling limbal stem cells may become hyperproliferative by stimulations, such as alterations in this anatomical site influenced by other factors, which can cause abnormal maturation of the conjunctival and corneal epithelium and lead to the formation of corneal intraepithelial neoplasia. Nevertheless, the etiology and pathogenesis of corneal intraepithelial neoplasia and ocular surface carcinoma remain elusive, because there is no appropriate animal model available to study the molecular and cellular mechanisms of this disease. Fibroblast growth factor-7 (FGF-7), a potent mitogen, enhances epithelial cell proliferation in various organs.4Finch PW Rubin JS Miki T Ron D Aaronson SA Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth.Science. 1989; 245: 752-755Crossref PubMed Scopus (818) Google Scholar, 5Rubin JS Osada H Finch PW Taylor WG Rudikoff S Aaronson SA Purification and characterization of a newly identified growth factor specific for epithelial cells.Proc Nat Acad Sci USA. 1989; 86: 802-806Crossref PubMed Scopus (738) Google Scholar, 6Rubin JS Bottaro DP Chedid M Miki T Ron D Cheon G Taylor WG Fortney E Sakata H Finch PW LaRochelle WJ Keratinocyte growth factor.Cell Biol Intern. 1995; 19: 399-412Crossref PubMed Scopus (264) Google Scholar The precise spatio-temporal expression of FGF-7 and the related polypeptide FGF-10 is important for governing ocular surface morphogenesis. FGF-7 and FGF-10 are secreted by mesenchymal cells, which bind with high affinity to the same FGF receptor 2 (FGFR2-IIIb) isoform expressed mainly by the epithelium.7Igarashi M Finch PW Aaronson SA Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7).J Biol Chem. 1998; 273: 13230-13235Crossref PubMed Scopus (271) Google Scholar In the cornea, expression of FGF-7 and its cognate receptor FGFR2-IIIb is higher in limbal stroma and epithelium, respectively, than in the central cornea, implicating that FGF-7 may promote limbal stem cell proliferation and participate in modulation of corneal epithelium renewal and homeostasis.8Li DQ Tseng SC Differential regulation of cytokine and receptor transcript expression in human corneal and limbal fibroblasts by epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor B, and interleukin-1 beta.Invest Ophthalmol Vis Sci. 1996; 37: 2068-2080PubMed Google Scholar, 9Li DQ Tseng SC Differential regulation of keratinocyte growth factor and hepatocyte growth factor/scatter factor by different cytokines in human corneal and limbal fibroblasts.J Cell Physiol. 1997; 172: 361-372Crossref PubMed Scopus (97) Google Scholar It has been reported that overexpression of FGF-7 or FGF-10 driven by αA-crystalline promoter, which is activated at mouse E11.5 before corneal epithelial differentiation, resulted in the suppression of cornea-type epithelial differentiation and the induction of ectopic lacrimal gland formation in the corneas of the transgenic mice.10Makarenkova HP Ito M Govindarajan V Faber SC Sun L McMahon G Overbeek PA Lang RA FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.Development. 2000; 127: 2563-2572Crossref PubMed Google Scholar, 11Govindarajan V Ito M Makarenkova HP Lang RA Overbeek PA Endogenous and ectopic gland induction by FGF-10.Dev Biol. 2000; 225: 188-200Crossref PubMed Scopus (72) Google Scholar, 12Lovicu FJ Kao WW Overbeek PA Ectopic gland induction by lens-specific expression of keratinocyte growth factor (FGF-7) in transgenic mice.Mech Dev. 1999; 88: 43-53Crossref PubMed Scopus (45) Google Scholar These results indicate that excess FGF-7 and FGF-10 are capable of altering epithelial fate decision during embryonic development. However, it remains unclear whether they are capable of exerting such an influence at a later stage when epithelial cells have committed to corneal epithelial differentiation. To answer this question, we developed a Krt12-rtTA/tet-O-FGF-7 double transgenic mouse line in which overexpression of FGF-7 in cornea is achieved by administering experimental mice doxycycline (Dox). In the present study, we show that excess FGF-7 induced by Dox in the Krt12-rtTA/tet-O-FGF-7 double transgenic mice beginning from embryonic day 0.5 to postnatal day 21 and beyond results in squamous cell carcinoma of the cornea, resembling OSSN in human. However, excess FGF-7 induced by Dox for up to 2 months in adult Krt12-rtTA/tet-O-FGF-7 double transgenic mice exhibits only corneal epithelial hyperplasia without tumor formation. The clinical manifestations of excess FGF-7 showed age-dependent lesions in the experimental mice. Experimental animals were housed under pathogen-free conditions in accordance with institutional guidelines. Animal care and use conformed to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati. Krt12-rtTA knock-in mice13Chikama T Hayashi Y Liu CY Terai N Terai K Kao CW Wang L Hayashi M Nishida T Sanford P Doestchman T Kao WW Characterization of tetracycline-inducible double transgenic Krt12rtTA/+/tet-O-LacZ mice.Invest Ophthalmol Vis Sci. 2005; 46: 1966-1972Crossref PubMed Scopus (34) Google Scholar were crossed to tet-O-FGF-7 mice14Tichelaar JW Lu W Whitsett JA Conditional expression of fibroblast growth factor-7 in the developing and mature lung.J Biol Chem. 2000; 275: 11858-11864Crossref PubMed Scopus (276) Google Scholar for the preparation of Krt12-rtTA/tetO-FGF7 double transgenic mice, which overexpress FGF-7 by corneal epithelium on induction of Dox. Transgenic mice were identified by genotyping of PCR using oligonucleotide primers specific for each transgene as follows. Krt12-rtTA knock-in allele was identified by PCR with the following oligonucleotide primers: forward Krt12-1 (primer 1), 5′-GTGTGTGCCTGCCATCCCATC-3′; Neo 781-803 (primer 2), 5′-CGCCTTCTTGACGAGTTCTTCTG-3′ for knock-in allele; and Krt12-1 primer and reverse Krt12-2 (primer 3), 5′-GAT CTG GGG TTG CAA TGA AGA C-3′ for wild-type allele. The primers for detecting tet-O-FGF-7 transgene in the transgenic mice were as follows: forward primer in CMV minimum promoter, 5′-GTCAGATCGCCTGGAGACGCC-3′; reverse primer in hFGF-7, 5′-AATTAGTTCTTTGAAGTTACAATCT-3′. PCR for Krt12-rtTA was performed by denaturation at 94°C for 5 minutes, 35 cycles of amplification (30 seconds at 94°C, 30 seconds at 64°C, and 45 seconds at 72°C), followed by a 5-minute final extension step at 72°C. PCR products were analyzed by agarose gel electrophoresis. Detection of tet-O-FGF-7 was identical except the annealing temperature was 54°C. To induce FGF-7 expression, mice were injected once intraperitoneally with Dox (80 μg/g body weight; Clontech Laboratories, Mountain View, CA) dissolved in PBS (pH 7.4) at a concentration of 10 mg/ml15Utomo AR Nikitin AY Lee WH Temporal, spatial, and cell type-specific control of Cre-mediated DNA recombination in transgenic mice.Nat Biotechnol. 1999; 17: 1091-1096Crossref PubMed Scopus (187) Google Scholar and fed Dox-chow (1 g/kg chow; Bioserv, Frenchtown, NJ). Control animals were fed regular chow. The following antibodies were used: rabbit anti-K12 antibody16Liu CY Zhu G Converse R Kao CW Nakamura H Tseng SC Mui MM Seyer J Justice MJ Stech ME Hansen GM Kao WW Characterization and chromosomal localization of the cornea-specific murine keratin gene Krt1.12.J Biol Chem. 1994; 269: 24627-24636Abstract Full Text PDF PubMed Google Scholar; rabbit anti-keratocan antibody17Liu CY Birk DE Hassell JR Kane B Kao WW Keratocan-deficient mice display alterations in corneal structure.J Biol Chem. 2003; 278: 21672-21677Crossref PubMed Scopus (146) Google Scholar; rabbit anti-VP-16 antibodies to detect rtTA (Clontech Laboratories); rabbit anti-hFGF7 (GeneTex, Inc., San Antonio, TX); rabbit anti-K14 and rabbit anti-Pax6 (Covance, Princeton, NJ); rabbit anti-phosphoERK1/2 (Neuromics, Inc., Edina, MN), monoclonal antibody 4A4 anti-p63 and monoclonal antibody anti-β-catenin (BD Biosciences); anti-E-cadherin (Chemicon, Inc., Temecula, CA) and 5-bromo-2′-deoxyuridine (BrdU) monoclonal antibody (Lab Vision, Corp., Fremont, CA). Secondary Alexa488, Alexa555, labeled antibodies were obtained from Molecular Probes, Inc. (Eugene, OR). Secondary horseradish peroxidase-conjugated antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). For Western blotting analysis, adult Krt12-rtTA/tet-O-FGF-7 mice (2 months old) were administrated with or without Dox for 2 weeks, and the neonatal Krt12-rtTA/tet-O-FGF-7 mice were administrated with or without Dox from embryonic day (E) 0.5 to postnatal day (P) 3. Eyes were then enucleated and incubated with Dispase II (0.5%) in PBS at 37°C for 2 hours to isolate corneal epithelial sheets. The corneal epithelial sheets were pooled and incubated with 1× cell lysis buffer containing 50 mmol/L Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Sigma, St. Louis, MO). Twenty micrograms of the total protein extract were separated on a SDS-PAGE and Western blotting analysis with rabbit anti-VP-16 antibodies and rabbit anti-hFGF7 followed by a goat anti-rabbit IgG (H+L)-alkaline phosphatase. Immune reactivity was visualized with Western blue (Promega, Madison, WI). Immunohistological analysis was performed to determine the consequences of excess FGF-7 in corneas of Krt12-rtTA/tet-O-FGF-7 mice and control single transgenic littermates. Excised eyes were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) at 4°C overnight and paraffin embedded. Five-micrometer sections were then mounted on Super Frost slides (Fisher Scientific, Pittsburgh, PA). The sections were de-paraffinized and hydrated in a graded ethanol series (95% and 75% ethanol and PBS for 3 minutes each). All incubations were performed at room temperature. Sections for immunofluorescence analysis were mounted (SlowFade Light Antifade kit; Molecular Probes) in the presence of 4-,6-diamidino-2-phenylindole, observed with an epifluorescence microscope (Axioscop2; Carl Zeiss, München-Hallbergmoos, Germany) and were photographed with a digital camera system (Axiocam, Carl Zeiss). The same fields were photographed under Nomarski optics, and the Nomarski and immunofluorescence images were overlaid. Transgenic mice bearing a single transgene, either Krt12-rtTA or tet-O-FGF-7, and noninduced Krt12-rtTA/tet-O-FGF-7 double transgenic mice had no anomaly in cornea or other tissues and survived normally in the vivarium.13Chikama T Hayashi Y Liu CY Terai N Terai K Kao CW Wang L Hayashi M Nishida T Sanford P Doestchman T Kao WW Characterization of tetracycline-inducible double transgenic Krt12rtTA/+/tet-O-LacZ mice.Invest Ophthalmol Vis Sci. 2005; 46: 1966-1972Crossref PubMed Scopus (34) Google Scholar, 14Tichelaar JW Lu W Whitsett JA Conditional expression of fibroblast growth factor-7 in the developing and mature lung.J Biol Chem. 2000; 275: 11858-11864Crossref PubMed Scopus (276) Google Scholar Adult Krt12-rtTA/tet-O-FGF-7 double transgenic and tet-O-FGF-7 single transgenic mice were treated with or without Dox for 2 weeks. Experimental animals were then sacrificed, and enucleated eyes were subjected to biochemical and histological analyses. Western blotting analysis of corneal epithelial extract showed compatible rtTA expression levels regardless of Dox treatment, but a large increase in FGF-7 only in Dox-treated Krt12-rtTA/tet-O-FGF-7 double transgenic mice (Figure 1A). None of Krt12-rtTA and tet-O-FGF-7 single transgenic mice fed Dox, showed elevated FGF-7 in the cornea (data not shown). The expression of FGFR2 receptor, which binds FGF-7 and FGF-10, was detected in corneal epithelium but did not seem to increase in response to FGF-7 overexpression (Figure 1B). The corneas remain transparent in all of the adult Krt12-rtTA/tet-O-FGF-7 mice treated with Dox (data not shown), however, histological examination of the enucleated eyes exhibited marked epithelial hyperplasia in the cornea (Figure 1, B and C). Corneal epithelium contained five to seven cell layers in untreated mouse but this increased to 15 to 18 cell layers in those treated with Dox (Figure 1, Figure 2). No corneal abnormality was observed in Krt12-rtTA or tet-O-FGF-7 single transgenic mice treated with Dox (data not shown). Incorporation of BrdU into DNA was used to estimate cell proliferation in the transgenic mice. As shown in Figure 1, C and D, Dox treatment caused an approximately 6.5-fold increase in BrdU incorporation by basal epithelial cells in the cornea and by some suprabasal cells in the peripheral cornea of Krt12-rtTA/tet-O-FGF-7 double transgenic mice. Because FGF/FGFR signaling transduction can phosphorylate and activate cytoplasmic ERK, we therefore examined the phospo-ERK by immunohistochmistry. As expected, nuclear localization of phospho-pERK1/2 was more prominent in Dox-induced compared with noninduced Krt12-rtTA/tet-O-FGF-7 double transgenic mice (Supplemental Figure 1A, see http://ajp.amjpathol.org). Likewise, β-catenin expression level was increased and was found in both membrane and nucleus (Supplemental Figure 1B, see http://ajp.amjpathol.org). In comparison, β-catenin was mostly found in the cytoplasm of control un-induced double transgenic Krt12-rtTA/tet-O-FGF-7 mice. The K12 expression pattern remained unchanged in all cell layers (basal through superficial layer) whether treated (Figure 2, A–C) or not treated by Dox (Figure 2, D–F). In contrast, K14 expression, normally restricted to the basal cell layer of stratified epithelium, was altered and detectable in nearly all cell layers in Dox-treated Krt12-rtTA/tet-O-FGF-7 double transgenic mice (Figure 2, compare G–I with J–L). Moreover, use of anti-K10 antibodies failed to detect the presence of this epidermal epithelium marker in the hyperplastic corneal epithelium, suggesting that no epithelial metaplasia was found in excess of FGF-7 (data not shown).Figure 2Dox-induced FGF-7 overexpression changes K14 but not K12 expression pattern. Immunohistofluorescent staining of corneas of the Krt12-rtTA/tet-O-FGF-7 mice administered with Dox chow (A–C and G–I) or Dox-free chow (D–F and J–L) with anti-K12 (A–F) or anti-K14 (G–L). K12 expression was detected in the full thickness of hyperplastic corneal epithelium in Dox-treated and of normal corneal epithelium in nontreated Krt12-rtTA/tet-O-FGF-7 mice. In contrast, K14 expression, which was only detected in the basal epithelium of the nontreated mice, was detected not only in the basal epithelium but also in suprabasal and superficial layers of the Dox-treated Krt12-rtTA/tet-O-FGF-7 mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effects of excess FGF-7 on corneal morphology in adult (older than 2 months) Krt12-rtTA/tet-O-FGF-7 double transgenic mice fed with Dox for various periods of time was assessed by histology and immunofluorescence staining (Figure 3). Without Dox induction (Figure 3, B, H, and N), corneal epithelial morphology and E-cadherin expression pattern of double transgenic mice were normal and indistinguishable from that of control tet-O-FGF-7 single transgenic mouse fed Dox for 14 days (Figure 3, A, G, and M). In contrast, corneal epithelium became hyperplasic after 24 hours Dox administration (Figure 3, C, I, and O) and increased to 15 to 18 cell layers at 7 days (Figure 3, D, J, and P) and 14 days (Figure 3, E, K, and Q), with altered E-cadherin expression pattern. Interestingly, 14 days after the termination of a 14-day Dox induction, the corneas of Krt12-rtTA/tet-O-FGF-7 double transgenic mice regained nearly normal corneal epithelial morphology and E-cadherin expression pattern (Figure 3, F, L, and R). Because K12 gene expression commences at E14.5,18Liu CY Zhu G Westerhausen-Larson A Converse RL Kao CW Sun TT Kao WW Cornea-specific expression of K12 keratin during mouse development.Curr Eye Res. 1993; 12: 963-974Crossref PubMed Scopus (109) Google Scholar, 19Tanifuji-Terai N Terai K Hayashi Y Chikama T Kao WW Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth.Invest Ophthalmol Vis Sci. 2006; 47: 545-551Crossref PubMed Scopus (71) Google Scholar systematic administration of Dox to pregnant Krt12-rtTA/tet-O-FGF-7 double transgenic females should lead to FGF-7 overexpression in the fetal corneal epithelium. To determine the effects of excess FGF-7 on corneas in utero, female Krt12-rtTA/tet-O-FGF-7 double transgenic mice were fed Dox from the time of conception (E0.5), and at least five embryos or pups were examined at various developmental stages: E16.5, P1, P3, and P21 as shown in Figure 4. Histological examination of the developing eyes from nontreated Krt12-rtTA/tet-O-FGF-7 double transgenic embryos at E16.5 showed that corneal epithelium consisted of two cell layers (Figure 5A), whereas Dox-treated embryos revealed marked hyperplasia (five to six layers) (Figure 5B). Immunohistochemical staining with anti-FGFR2 showed that nearly the entire corneal epithelium was FGFR2 positive (Figure 5C), and Dox-induced hyperplastic epithelium retains high expression of FGFR2 (Figure 5D), suggesting it continues to respond to the excess FGF-7. When BrdU labeling was compared between corneal sections in the Dox-treated (Figure 5F) and nontreated Krt12-rtTA/tet-O-FGF-7 mice (Figure 5E), quantitation of cell proliferation revealed that there was an approximately 9-fold increase of BrdU uptake (Figure 5G) in the Dox-treated mice. K14 keratin, which is normally expressed only in the basal epithelium, was detected in the entire hyperproliferative epithelium (Figure 5, J and K), but the corneal differentiation marker K12 was found only in the superficial layer (Figure 5, H and I).Figure 5Dox-induced epithelial hyperplasia in the embryonic cornea. Histological examination of Krt12-rtTA/tet-O-FGF-7 embryos (E16.5) with (B, D, F, I, and K) or without (A, C, E, H, and J) exposure to Dox through pregnant mothers from E0.5. Dox treatment induced corneal epithelial hyperplasia (B). Immunohistochemical staining with anti-FGFR2 (C and D) showed that nearly the entire epithelium is FGFR-positive (C); likewise, hyperplastic epithelium retains high expression of FGFR2 (D). Hyperplastic epithelium responds to FGF-7 with increased BrdU uptake (F) compared with noninduced embryos (E). A histogram shows that there is a 9.1-fold increase of BrdU uptake per corneal sections in the Dox-treated (filled bar) compared with the nontreated (open bar) Krt12-rtTA/tet-O-FGF-7 mice (G). Immunohistochemical stainings with anti-K12 (H and I) and anti-K14 (J and K) were used to characterize the epithelial differentiation status. Nearly the entire hyperplastic epithelium is K14-positive (K), but only the superficial layer exhibits K12-positive staining (I). Scale bars = 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) At P1, corneal epithelium of Krt12-rtTA/tet-O-FGF-7 double transgenic pups that had been exposed to Dox from E0.5 had around 20 cell layers, 10 times more than that of single tet-O-FGF-7 transgenic littermates and of age-matched noninduced Krt12-rtTA/tet-O-FGF-7 double transgenic pups (Figure 6). K12 was detected in the superficial layer but absent in the basal and suprabasal layers (Figure 6, F and G). However, K14 was expressed throughout the entire epithelial layer (Figure 6, H and I). It is of interest to note that K12- and K14-negative hyperproliferative nodules existed in the limbal basal region of the Dox-treated Krt12-rtTA/tet-O-FGF-7 double transgenic eyes as shown in Figure 6, G and I. This observation was extended in the postnatal day 3, Krt12-rtTA/tet-O-FGF-7 double transgenic pups exposed to Dox from E0.5 (Figure 7), in that K12-negative (Figure 7E) and K14-negative (Figure 7F) hyperproliferative nodules labeled by BrdU were present across the entire corneal epithelium (Figure 7D). Although hyperplasia is prominent in the epithelium, corneal and limbal stroma remained relatively unaffected by excess FGF-7 present in the epithelium, because the expression pattern of keratocan, a keratocyte-specific marker, was not altered (Figure 6, J–M).Figure 7Dox-induced epithelial hyperplasia and ectopic gland invasion in the neonatal cornea. Krt12-rtTA/tet-O-FGF-7 mouse exposed to Dox through pregnant mothers from E0.5 showed hyperproliferative epithelium at postnatal day 3 (B) compared with age-matched noninduced Krt12-rtTA/tet-O-FGF-7 mouse (A). C: Western blotting analyses of 20 μg of total protein extract isolated from corneal epithelial sheets showed that hFGF-7 was dramatically up-regulated in the corneal epithelium of Dox-treated (lane 2) compared with nontreated age-matched mice (lane 1). gapdh served as a reference of protein loading. At this stage, it was clear that the stromal-epithelial boundary was not preserved due to the corneal invasion by the ectopic glandular cell mass (see Figure 6), which was extremely hyperproliferative as revealed by strong BrdU immunoreactivity (D) and K12 (E) and K14 (F) negativity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Krt12-rtTA/tet-O-FGF-7 double transgenic mice under continued Dox induction from E0.5 to P21 manifested thickened mulberry-like gelatinous lesions with gray intraepithelial plaques and tufts of vessels in the corneas, resembling human corneal intraepithelial neoplasia (Figure 8B). Histological examination revealed that excess FGF-7 induced a papilloma-like tumor with neovascularization and stromal invasions into the corneal epithelium (Figure 8, C and D). A differentiated glandular cell mass was apparent in the limbal basal region (Figure 8E, inset). Immunohistochemical analyses showed that the hyperplastic epithelial cells stained heavily for Pax-6 (Figure 9, A–D) in comparison with a single basal layer staining in noninduced corneas (Figure 9, E–H). Similarly, overexpression of ΔNp63 was observed in almost all epithelial cells (Figure 9, K and L), whereas in control un-induced double transgenic mice, ΔNp63 was observed only in the basal corneal epithelial cells (Figure 9, I and J). Unlike Dox-treated adult Krt12-rtTA/tet-O-FGF-7 mice in which the epithelial hyperplasia is reversible when Dox is removed, corneal tumors in Krt12-rtTA/tet-O-FGF-7 mice induced from E0.5 to P21 did not completely regress, and normal corneal morphology did not return even 60 days after Dox withdrawal (Figure 10).Figure 9Corneal intraepithelial neoplasia induced by Dox expressed both Pax-6 and p63 progenitor markers. Krt12-rtTA/tet-O-FGF-7 mice exposed to Dox through pregnant mothers from E0.5 exhibited corneal intraepithelial neoplasia at postnatal day 21 (A, B, C, D, K, and L) compared with age-matched noninduced Krt12-rtTA/tet-O-FGF-7 (E–J). Immunofluorescent staining was performed to detect Pax-6 (green) and counterstained with 4-,6-diamidino-2-phenylindole (blue) (A–H). Immunohistochemical staining was performed to detect p63 (I–L). Both Pax-6 and p63 were detected throughout the entire hyperplastic epithelium in the Dox-induced Krt12-rtTA/tet-O-FGF-7 mice (A, B, C, D, K, and L), whereas they were only detected in the basal epithelial layer in the noninduced Krt12-rtTA/tet-O-FGF-7 (E–J).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Reversibility of corneal intraepithelial neoplasia. Photographs of both left (OS) and right (OD) eyes were taken from Krt12-rtTA/tet-O-FGF-7 mice exposed to Dox from E0.5-P21 followed by Dox-free diet for 0 (A and F), 15 (B and G), 30 (C and H), 45 (D and I), and 60 (E and J) days.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine the hypothesis that continuous growth stimulation may result in the alteration of corneal epithelial homeostasis that can lead to corneal pathology, such as OSSN, we have established cornea-specific and Dox-inducible overexpression of FGF-7 in transgenic mouse lines, namely, Kerapr-rtTA/tet-O-FGF-720Hayashi M Hayashi Y Liu CY Tichelaar JW Kao WW Over expression of FGF7 enhances cell proliferation but fails to cause pathology in corneal epithelium of Kerapr-rtTA/FGF7 bitransgenic mice.Mol Vis. 2005; 11: 201-207PubMed Google Scholar and Krt12-rtTA/tet-O-FGF-7 (this study). Unlike Dox-treated Krt12-rtTA/tet-O-FGF-7 mice, which exhibited severe corneal phenotypes, Kerapr-rtTA/tet-O-FGF-7 double transgenic mice exposure to Dox exhibited a subtle phenotypic change of epithelial hyperproliferation without significant corneal pathology.20Hayashi M Hayashi Y Liu CY Tichelaar JW Kao WW Over expression of FGF7 enhan
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