|Molecular Vision 2005;
Received 2 February 2005 | Accepted 15 March 2005 | Published 16 March 2005
Over expression of FGF7 enhances cell proliferation but fails to cause pathology in corneal epithelium of Kerapr-rtTA/FGF7 bitransgenic mice
Miyuki Hayashi,1 Yasuhito Hayashi,1 Chia-Yang
Liu,1 Jay W. Tichelaar,2 Winston
Departments of 1Ophthalmology and 2Environmental Health, University of Cincinnati, Cincinnati, OH
Correspondence to: Winston W.-Y. Kao, Ph.D., Department of Ophthalmology, University of Cincinnati, 3223 Eden Avenue, Cincinnati, OH, 45267-0527; Phone: (513) 558-2802; FAX: (513) 558-3108; email: Winston.Kao@uc.edu
Purpose: Available evidence suggests that fibroblast growth factor 7 (FGF7, also known as keratinocyte growth factor, KGF) serves as a paracrine growth factor modulating corneal epithelial cell proliferation. In the present study, we used a binary inducible transgenic mouse model to examine the role of FGF7 on corneal epithelium proliferation.
Methods: A keratocyte specific 3.2 kb murine keratocan promoter (Kerapr) was used to prepare Kerapr-rtTA transgenic (Kr) mice that constitutively overexpress reverse tetracycline transcription activator (rtTA) by cornea stromal keratocytes. The Kr mice were crossed with tet-O-FGF7 mice to produce Kr/tet-O-FGF7 bitransgenic mice. Expression of human FGF7 (hFGF7) was induced by the administration of doxycycline via intraperitoneal injection and/or feeding mice doxycycline in drinking water and chow. Overexpression of hFGF7 was confirmed by RT-PCR and western blot. BrdU incorporation was used to determine cell proliferation.
Results: The rtTA mRNA and protein were constitutively expressed by the cornea with or without doxycycline induction, whereas hFGF7 was detected only in Kr/tet-O-FGF7 bitransgenic mice upon induction by doxycycline. Examination of induction kinetics in adult Kr/tet-O-FGF7 bitransgenic mice after a single intraperitoneal injection of doxycycline revealed that hFGF7 mRNA expression was detected 12 h after doxycycline administration, peaked at 36 h, was sustained up to 48 h, and declined thereafter. The elevated level of hFGF7 expression coincided with hyperproliferation of corneal epithelial cells. In bitransgenic mice, the number of BrdU labeled cells increased after 36 and 48 h of transgene induction compared to controls of noninduced bitransgenic or doxycycline treated single transgenic mice. The BrdU labeling index was 33±9.2 positive cells per corneal section for Kr/tet-O-FGF7 bitransgenic mice and 25±9.3 for tet-O-FGF7 single transgenic mice at 36 h post-doxycycline treatment. However, the excess FGF7 driven by doxycycline induction did not produce severe perturbation of corneal epithelium homeostasis.
Conclusions: Our results demonstrate that the doxycycline inducible system is effective in regulating transgene expression in corneal stroma of Kr/tet-O-FGF7 bitransgenic mice. However, the development of pathology resulting from the overexpression of transgenes may depend on whether the amount of transgene product present is sufficient to alter the homeostasis of the targeted tissues.
Transgenesis via microinjection of cloned DNA into fertilized mouse eggs was first accomplished nearly simultaneously in the laboratories of Brinster, Costantini, Ruddle, Mintz, and Wagner (for review see [1,2]). Since then, transgenic animal technology has been applied in many situations. Altered phenotypes resulting from transgene expression have demonstrated that introduced genes can exert profound effects on animal physiology. While many transgenic mouse models have proven useful for the identification of growth factors and transcription factors that are important for mouse ocular tissue morphogenesis, their use in further studies is often less successful because of systemic and/or congenital defects resulting from non-tissue specific and constitutive transgene expression. Thus, it is difficult, if not impossible, to determine their roles in ocular surface tissue morphogenesis . Therefore, there is a need for generating mouse lines in which the expression of a transgene is modulated in a specific spatial-temporal manner [4,5].
Fibroblast growth factor 7 (FGF7), a member of the FGF family, is a mesenchyme derived mitogen for epithelial cells. FGF receptors are expressed by epithelial cells of numerous tissues, but FGF7 is produced in adjacent stromal cells. Thus FGF7 is thought to act as a paracrine factor in mediating epithelial cell behavior [6-9]. Recently, transgenic mice that overexpress FGF7, using a lens specific αA-crystalline promoter (αAcpr) have been created. The transgenic mice overexpressing FGF7 exhibit adversely affected ocular organogenesis, and have an abnormal K12 expression pattern during development . The most striking phenotypic development of transgenic mice overexpressing FGF7 by αAcpr was the hyper proliferation of embryonic corneal epithelial cells and their subsequent differentiation into functional lacrimal gland-like tissues. This indicates that stimulation of the FGF7 receptor early in development, in surface ectoderm normally destined to form corneal epithelium, is sufficient to alter the fate of these cells. Furthermore, this suggests that the correct spatial and temporal expression of FGFs plays a critical role in normal lacrimal gland induction.
In the present study, a keratocyte specific 3.2 kb murine keratocan promoter (Kerapr) that specifically directed LacZ reporter gene expression in a pattern similar to that of the endogenous keratocan gene (Kera) was used . The keratocyte specific promoter was used to generate a transgenic mouse model that constitutively directs expression of the reverse tetracycline transactivator protein (rtTA) in keratocytes. The Kerapr-rtTAKr transgenic mice were crossbred with tet-O-FGF7 transgenic mice  to produce bitransgenic (Kr/tet-O-FGF7) offspring, allowing for the expression of the FGF7 transgene following doxycycline treatment. The overexpression of FGF7 resulted in enhanced epithelial cell proliferation, although alterations in corneal pathology were limited.
Construction of pKerapr3.2-rtTA-BpA plasmid
An EcoRI-BamHI fragment (about 1 kb) containing the rtTA coding sequence was excised from pTet-ON vector (Clontech, Palo Alto, CA) and subcloned into the EcoRI and BamH I digested pBKCMV vector (Stratagene La Jolla, CA) to generate the pBKCMV-rtTA plasmid. The Kerapr3.2-MCS-BpA (keratocan promoter-bovine growth factor polyadenylation signal) vector contains ClaI and SalI sites available for subcloning. The N-terminal portion of rtTA (about 800 bp) obtained from pBKCMV-rtTA plasmid DNA by ClaI and SalI digestion was cloned into the Kerapr3.2-MCS-BpA vector. The resulting plasmid clone was designated as pKerapr3.2-rtTAΔC-BpA. Finally, the SalI fragment (about 200 bp) of pBKCMV-rtTA which encodes the C-terminal portion of the rtTA was cloned into the SalI site of pKerapr3.2-rtTAΔC-BpA vector. The resulting plasmid was designated as pKerapr3.2-rtTA-BpA. The fidelity of the rtTA construct was confirmed by DNA sequencing.
Generation and genotyping of Kerapr3.2-rtTA transgenic mice
The Kerapr-rtTA-BpA transgene (4.9 kb) was excised from the pKerapr3.2-rtTA-BpA with NotI and KpnI and microinjected into pronuclei of fertilized mouse oocytes by the Transgenic Core Facility at the University of Texas at San Antonio. Transgenic founders were identified by Southern hybridization of mouse tail DNA digested with EcoRI and BamHI using a 32P labeled rtTA probe.
Transgenic mice were subjected to genotyping by polymerase chain reaction (PCR) with tail DNA using primer pairs for rtTA, 5' primer in Kerapr, 5'-CCT AAC ACC AGC CAC AGG ACT, and 3' primer in rtTA, 5'-CGT ACT CGT CAA TTC CAA GGG C; and hFGF-7, 5' primer in CMV minimum promoter, 5'-GTC AGA TCG CCT GGA GAC GCC, 3' primer in hFGF7, AAT TAG TTC TTT GAA GTT ACA ATC T, using the following conditions: 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s, and 72 °C for 5 min. PCR products were analyzed by electrophoresis in 1% agarose gels.
A Kerapr-rtTA-BpA minigene construct (Figure 1A) was used to prepare transgenic mouse lines (Kr) that constitutively overexpress rtTA by cornea stromal keratocytes. Female Kr mice were crossed with male tet-O-FGF7 transgenic mice  to obtain bitransgenic Kr/tet-O-FGF7 mice. Single transgenic Kr or tet-O-FGF7 littermates were used as control. The mice were bred and housed in the Laboratory Animal Medical Services of the University of Cincinnati. Animal protocols adhere to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the IACUC of the University of Cincinnati. Eight-week-old bitransgenic and single transgenic mice were injected intraperitoneally with doxycycline (80 μg/g body weight; Clontech Laboratories) dissolved in PBS (pH 7.4) at a concentration of 10 mg/ml . Two h prior to sacrificing the experimental mice by CO2 asphyxia and cervical dislocation, BrdU (200 μg/g body weight) was injected intraperitoneally as previously reported . In another series of experiments, induction was continued for up to 2 months by administering doxycyline in drinking water (20 mg/ml) and/or chow (1 g/Kg chow).
After various periods of doxycycline induction (e.g., 0, 12, 24, and 48 h) experimental mice were sacrificed and total RNA was isolated from corneas using Trizol Reagent (GIBCO, Grand Island, NY). The total RNA isolated was treated with RNase free DNAse (Promega, Madison, WI), cDNA was synthesized by AMV Reverse Transcriptase (Promega) and subjected to RT-PCR for detecting hFGF7 and rtTA mRNAs. The following primer pairs were used: hFGF7; 5' hFGF-7 primer, 5'-CTA CAG ATC ATG CTT TCA CAT TA; 3'hFGF7primer, 5'-AAT TAG TTC TTT GAA GTT ACA ATC T; 5'rtTAprimer, 5'-CTT AAA TGT GAA AGT GGG TCC GCG; 3' rtTA primer, 5'-CGT ACT CGT CAA TTC CAA GGG C. PCR amplification was performed as described above.
Western blotting analysis
Mice were sacrificed 0, 12, 24, 36, and 48 h after doxycycline treatment (single intraperitoneally injected). Corneas excised from mice were homogenized in RIPA buffer containing 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, protease inhibitor cocktail (Sigma, St. Louis, MO), and incubated at 4 °C for 24 h to adequately extract soluble proteins. The homogenate was centrifuged at 10,000x g, and the supernatant was stored frozen until use. Corneal extracts were separated by gradient polyacrylamide gel electrophoresis (4-15%; Bio-Rad Laboratories, Hercules, CA) and transferred to a PVDF Immobilon-P membrane (Millipore Corp., Bedford, MA) as described previously . The membrane was probed with primary polyclonal anti-VP-16 antibodies that cross-reacted to rtTA (Clontech Laboratories), rabbit anti-hFGF7, and goat anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA) according to the procedures recommended by the manufacturers, followed by a goat anti-rabbit IgG (H+L)-alkaline phosphatase (AP; Southern Biotechnology, Birmingham, AL) and goat anti-rabbit Ig (H+L)-horse radish peroxidase (HRP; Southern Biotechnology) and donkey anti-goat IgG HRP (Santa Cruz Biotechnology), respectively. Immune reactivity was visualized with Sigma FastTM (Sigma) or ECL plus Western Blotting Detection (Amersham Biosciences, Piscataway, NJ) reagents using procedures recommended by the manufacturer (Sigma), respectively.
Experimental mice were intraperitoneally injected with BrdU (8 mg/ml in PBS, 200 μg/g body weight) 2 h prior to sacrifice. Eyes enucleated from experimental mice were fixed in 4% paraformaldehyde and 0.1 M phosphate buffer pH 7.4 (PB) at 4 °C overnight and embedded in paraffin. Sections (5 μm) were used for immunohistochemistry with anti-BrdU antibody (Neo Markers, Fremont, CA) followed by horse anti-mouse IgG (H+L) AP (Vector Laboratories), and visualized by 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP; Roche, Indianapolis, IN) and 4-Nitroblue tetrazolium chloride (NBT; Roche). BrdU positive corneal epithelial cells were counted from 6 sections per cornea. Four to six animals were used in each experimental condition. Statistical significance was assessed by the unpaired t-test.
To investigate the change of corneal morphology, single (Kr, n=5; tet-O-FGF7, n=5) and bitransgenic (Kr/tet-O-FGF7, n=5) mice were fed doxycycline water (20 mg/ml) and intraperitoneally injected with doxycycline (once a day) for one week. Water bottles were covered with aluminum foil to protect the antibiotic from light. Eye tissue sections from experimental mice were stained with hematoxylin and eosin or underwent immunohistochemistry with anti-keratin 12 antibody as described previously . To detect anti-keratin 12, goat anti-rabbit IgG-AP (Southern Biotechnology) followed by BCIP and NBT were used. Bitransgenic mice fed water without doxycycline and not intraperitoneally injected were used as negative controls (n=4).
We also examined the mice treated long term with doxycycline water and food. Adult bitransgenic (Kr/tet-O-FGF7, n = 4) and single transgenic mice (Kr, n=3 and tet-O-FGF7, n = 1) were fed 2% doxycycline water and doxycycline chow (1 g/kg chow) for 2 months. Mouse eyes were subjected to hematoxylin and eosin staining and to anti-BrdU staining.
Corneal wound healing
Experimental mice were induced to express the hFGF7 transgene by an intraperitoneal injection of doxycycline 24 h prior to corneal epithelium debridement created with an Algerbrush II® (The Alger Company, Lago Vista, TX). Ten bitransgenic (Kr/tet-O-FGF7) and six single transgenic mice (Kr and/or tet-O-FGF7) were used in the experiment. Photographs of fluorescein stained eyes were taken at 12 and 24 h after debridement to evaluate healing of the epithelium as described previously .
The expression of rtTA by corneal cells of Kr mice
Four transgenic founder mice (Kr23, 31, 33, and 34) were identified by Southern hybridization (Figure 1B) following microinjection of the Kerapr3.2-rtTA-Bpa minigene (Figure 1A). All four founder mice transmitted the transgene to their offspring as determined by PCR genotyping of tail DNA (data not shown). The expression of rtTA protein by corneal keratocytes of transgenic mice was determined by western blot analysis. Only line Kr23 constitutively expressed the reverse tetracycline responsive transactivator (rtTA) under the control of the Kerapr (Figure 1C). Western blots of corneal extracts from line Kr23 had a 37 kDa rtTA band and an uncharacterized cellular protein with a molecular weight at 70 kDa that was also detected in non transgenic littermates. The presence of this 70 kDa band in tissue homogenates has previously been described by the manufacturer (Figure 1C). The rtTA expressing mouse line was then crossed with the tet-O-FGF7 transgenic mouse described previously . The other lines were not further examined.
Expression of FGF7 by doxycycline induction
Expression of hFGF7 in corneal tissue from bitransgenic Kr/tet-O-FGF7 mice was detected by RT-PCR at 12, 24, and 48 h after a single intraperitoneally injected administration of doxycycline, whereas mRNA of rtTA was detected in all corneas with or without induction (Figure 2A). To verify the induction of FGF7 protein by doxycycline treatment, western blot analysis was performed with corneal extracts from transgenic mice treated with doxycycline. hFGF7 protein was detected in corneas of compound transgenic mice that were induced with doxycycline for 12 to 48 h. The amount of FGF7 in corneas of compound transgenic mice peaked at 36 h after induction. In single transgenic Kr and tet-O-FGF7 mice, a small amount of FGF7 was detected, similar to that of un-induced bitransgenic mice (0 h). As the FGF7 antibody cross-reacts with both mouse and human FGF7, this likely represents the level of endogenous mouse FGF7 in cornea (Figure 2B).
To examine if excess FGF7 stimulated corneal epithelial cell proliferation, BrdU incorporation by epithelial cells was determined. BrdU positive epithelial cells were located in the peripheral cornea and limbus of un-induced bitransgenic and/or single transgenic mice (Figure 3B). At 36 or 48 h after an intraperitoneal injection of doxycycline, more BrdU labeled cells were observed in the center of the corneal basal cell layer of bitransgenic mice (Figure 3A). The number of BrdU labeled corneal epithelial cells in the bitransgenic mice following induction for 36 h (34.8±9.2 cells/field, n=24) or 48 h (33.3±7.5 cells/field, n=24) was increased significantly (p<0.01) compared to single transgenic littermates (tet-O-FGF7, 25.3±9.3 cells/field, n=30; Kr, 25.5±7.8 cells/field, n=36; Figure 4). In bitransgenic mice, the number of BrdU positive cells increased 36 or 48 h after doxycycline treatment, then returned to baseline at 72 h after doxycycline treatment.
Corneal morphology in steady state and during wound healing
In the cornea of 1-week doxycycline treated (intraperitoneally injected) bitransgenic mice, similar hyperproliferative epithelium was observed in the basal epithelial layer as detected by BrdU incorporation (data not shown). The corneas of bitransgenic mice that were induced for a prolonged period of time (up to 2 months) by providing increased amounts of doxycycline in drinking water (20 mg/ml) and chow (1 g/kg) also showed an increase of BrdU labeled epithelial cells compared with single transgenic mice. After 2 months of treament, the number of BrdU labeled corneal epithelial cells in bitransgenic mice (38.1±9.1, n=24) increased significantly (p<0.01) compared to that of single transgenic mice (26.4±8.8, n=24). There was no difference in the expression pattern of keratin 12 between doxycycline treated and untreated bitransgenic mice (data not shown). The number of epithelial cell layers remained the same among all groups of experimental mice. Taken together, the observations suggest that the level of excess FGF7 induced by doxycycline in bitransgenic mice was insufficient to cause perturbation of corneal epithelium homeostasis.
We also examined whether the expression of FGF7 had an impact on the healing of corneal epithelium. FGF7 transgene expression was induced by doxycycline 24 h prior to epithelium debridement. There was no significant difference in the rate of wound healing (p<0.05) between induced bitransgenic and single transgenic mice 12 and 24 h after epithelium debridement (remaining epithelial defects at 12 h: Kr/tet-O-FGF7, 62%±22% [n=10], Kr, 57%±22% [n=6]; at 24 h: Kr/tet-O-FGF7, 22%±15% [n=10], Kr, 21%±11% [n=6]) as determined by fluorescein staining .
We have prepared a bitransgenic mouse line that expresses hFGF7 in stromal keratocytes upon induction by doxycycline. The presence of excess FGF7 caused corneal epithelium hyperproliferation, but failed to produce obvious pathology in the induced bitransgenic mice. Members of the FGF gene family function in mediating epithelial-mesenchymal interactions and play important roles in early pattern formation during embryonic development [17-20]. Expression of these genes at early and late developmental stages indicate that they play important roles as signaling molecules throughout the life span of the organism [21-24]. The FGFs constitute a large family of at least 23 distinct polypeptide growth factors (FGF1 to FGF23), which share sequence homology [25-28]. The FGFs mediate their pleiotropic actions by binding and activating high affinity membrane-spanning receptors that form a distinct subgroup of the receptor tyrosine kinase family . FGF7 is a mesenchyme derived mitogen for epithelial cells. FGF receptors are expressed by the epithelial cells of numerous tissues. Thus, FGF7 is thought to act as a paracrine factor in mediating epithelial cell behavior [6-9,30].
It had been demonstrated that overexpression of FGF7 caused severe pathology in lung , cornea , and lens morphogenesis . However, in this study Kerapr-rtTA/tet-O-FGF7 bitransgenic mice exhibited a subtle phenotypic change of epithelial hyperproliferation without perturbing cornea-type epithelial differentiation as assessed by keratin 12 immunostaining. This is distinct from the severe phenotype observed in mice overexpressing FGF7 driven by a lens specific αA-crystalline promoter . The corneal epithelium of bitransgenic mice induced by doxycycline had normal epithelial stratification and normal healing after epithelium debridement. The subtle phenotype observed might be due to a level of FGF7 protein that was not sufficient to perturb corneal epithelium homeostasis. The presence of extracellular matrix in stroma and the basement membrane may preclude sufficient amounts of FGF7 from reaching the epithelium. Alternatively, due to the avascular characteristic of the cornea, the concentration of doxycycline in stroma may be insufficient to activate the tet-O-FGF7 transgene. These possibilities are consistent with our recent observation that over expression of FGF7 by corneal epithelial cells of bitransgenic K12rtTA/+/tet-O-FGF7 mice caused severe corneal epithelial dysplasia upon doxycycline treatment (unpublished). It is likely the doxycycline derived from tears may be readily accessible to epithelial cells, but not stromal keratocytes. In conclusion, the use of the tet-on system to overexpress an FGF7 transgene in corneal keratocytes did not result in dramatic corneal pathology. However, the development of the keratocyte specific inducible transgenic model may be useful for the study of other transgenes in this cell type.
The studies were in part supported by grants from NIH EY13755 and EY12486, Research to Prevent Blindness, Inc. (RPB), Ohio Lions Eye Research Foundation. WWYK is a RPB Senior Investigator in 2003. CYL is a recipient of the RPB Olga Weiss Scholarship.
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