Molecular Vision 2001; 7:20-26 <http://www.molvis.org/molvis/v7/a4/> Received 22 August 2000 | Accepted 2 February 2001 | Published 8 February 2001

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Characterization of Corn1 mice: Alteration of epithelial and stromal cell gene expression

I-Jong Wang,¹ Candace W.-C. Kao,¹ Chia-Yang Liu,¹ Shizuya Saika,¹ Patsy M. Nishina,² John P. Sundberg,² Richard S. Smith,² Winston W.-Y. Kao¹
 
 

¹Department of Ophthalmology, University of Cincinnati, Cincinnati, OH; ²The Jackson Laboratory, Bar Harbor, ME

Correspondence to: Winston W.-Y. Kao, Department of Ophthalmology, University of Cincinnati, Eden and Bethesda Avenue, Cincinnati, OH, 45267-0527; Phone: (513) 558-2802; FAX: (513) 558-3108; email: winston.kao@uc.edu

Abstract

Purpose: Corn1 is an autosomal recessive mutation characterized by corneal epithelial hyperplasia and stromal neovascularization. The aim of the present study is to examine the expression patterns of specific epithelial and stromal proteins in corn/corn1 mutant mice.

Methods: Immunohistochemistry with antibodies directed against keratins 1, 4, 5, 12, and 14 as well as loricrin, filaggrin, and involucrin were performed in corn1/corn1 and wild type, A.By/SnJ strain, mice at 4 weeks of age. Western blot hybridization was performed to confirm the presence of involucrin in corneas. In situ and northern blot hybridization were used to evaluate the expression of keratin 12, lumican, and keratocan in these mice.

Results: In corn1/corn1 mice, focal areas of corneal epithelial hyperplasia alternate with epithelium with normal appearance. Both regions of normal and hyperplastic corneal epithelium were labeled by anti-keratin 12 antibodies through all corneal epithelial layers. The anti-keratin 14 antibody only labeled the basal cell layer in normal epithelial areas, whereas it labeled both basal and suprabasal cell layers in hyperplastic areas. In wild type mice, anti-keratin 12 antibodies labeled all corneal epithelial layers, whereas anti-keratin 14 labeled the basal corneal epithelial cells only. Positive staining by anti-involucrin antibody was demonstrated in the basal corneal epithelial layer of wild type mice and normal areas of corn1/corn1 mice. Similarly, as observed with anti-keratin 14 antibody, the anti-involucrin antibody labeled both basal and suprabasal cell layers of hyperplastic corneal epithelium of corn1/corn1 mice. Antibodies against keratin 1, keratin 4, loricrin, and fillagrin did not label the corneas of wild type mice or corn1/corn1 mice. Northern hybridization indicated that the expressions of keratocan and lumican mRNA levels were up regulated in corn1/corn1 mice, but keratin 12 mRNA remained similar to that of the wild type mice. In situ hybridization revealed that the lumican mRNA was detected in epithelial and stromal cells of corn1/corn1 mice, whereas keratocan mRNA was only detected in stromal cells.

Conclusions: Hyperproliferative epithelial cells of corn1/corn1 mice have increased levels of expression of keratin 14 and involucrin, but do not exhibit the phenotypical characteristics of cornification. These observations indicate that factors associated with the phenotypes of corn1/corn1 mice do not alter the cornea-type epithelial differentiation of keratin 12 expression, but cause aberrant expression of lumican by corneal epithelial cells.

Introduction

Like other squamous epithelial cells that express specific keratin intermediate filaments in a tissue-specific manner, the corneal epithelium expresses the keratin 3 and 12 pair in all cell layers as distinct differentiation markers [1-4]. Normal corneal and conjunctival epithelia do not produce a cornified envelope, a characteristic that differs from other stratified epithelia, e.g., epidermal epithelium [5]. However, transition of non-cornified, stratified ocular surface epithelium into hyperplastic cornified epithelium may occur during squamous metaplasia [6-9].

It is noteworthy that epithelial hyperplasia and neovascularization are frequently associated with the pathogenesis of many ocular diseases characterized by cornification and by alterations in patterns of corneal epithelium-specific keratin expression, e.g., keratitis sicca, xerophthalmia, chemical and thermal burns, ocular cicatricial pemphigoid, and Stevens-Johnson syndrome [6-9]. Therefore, alteration of gene expression of corneal epithelial cells caused by factor(s) of corn1/corn1 mice may subsequently induce stroma neovascularization or vice versa. It has been suggested that bi-directional interactions and signaling mediated by growth factors and cytokines between mesenchyme and epithelium are essential for morphogenesis, during development and maintenance of homeostasis [10,11]. For example, it has been recently demonstrated that excess FGF-7 in ocular tissues of mouse embryos perturbs the differentiation of ocular surface ectoderm that normally gives rise to the formation of corneal and conjunctival epithelium and epithelium of exocrine glands, such as the lacrimal gland, instead the corneal surface is covered by conjunctival epithelium [12].

The corn1 phenotype is caused by an autosomal recessive mutation on mouse chromosome 2 [13]. Corneal epithelial hyperplasia has been noted at 12 days after birth before eyes open in homozygous corn1/corn1 mice and persists through life, whereas neovascularization begins shortly after eyes open, and progresses during the first 2 months of life and never regresses. Actually, there is evidence that there is irregular epithelial thickening in corneas of corn1/corn1 mice by about one week of age with light microscopy. The epithelial cells are highly proliferative and are characterized by increased DNA synthesis. The gene of corn1/corn1 has not been identified, yet. Thus, the molecular and cellular mechanisms of corneal neovascularization and epithelial hyperplasia observed in corn1/corn1 mice remain elusive. It is also not known whether factors associated with vascularization and epithelial hyperplasia in corn1/corn1 mice may disrupt the expression patterns of cornea-specific genes, i.e., keratin12 and keratocan, by corneal epithelial cells and keratocytes, respectively [14,15]. To investigate specific cellular markers of corn1/corn1 corneas, we examined the expression patterns of specific proteins in corneal epithelial and stromal layers of corn1/corn1 mice by immunohistochemistry with antibodies directed against components of the cornified envelope and keratins. In situ and northern blot hybridization were also used to evaluate the expression of keratin 12, lumican, and keratocan mRNAs. Our results indicate that the epithelial hyperplasia found in corn1/corn1 mice is characterized by altered involucrin and keratin 14 expression, but does not lead to complete cornification in ocular surface epithelium.

Methods

Animals

Twelve four-week old homozygous corn1/corn1 A.By/SnJ mice were used in this study. Six age-matched mice of the wild type A.By/SnJ strain were used as controls. The animal protocol adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati.

Immunohistochemistry

Eyes enucleated immediately after euthanasia by CO₂ asphyxia and fixed in 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) at 4 °C overnight were embedded in paraffin. Five mm-thick paraffin sections were incubated with specific antibodies by procedures as previously described [14,16]. Rabbit anti-peptide antibodies (BabCo, Berkeley, CA) specific for mouse involucrin, loricrin, keratin1 and fillagrin (all diluted 1:500) were used for the detection of the cornified envelope. Anti-keratin 4 and anti-keratin 14 antibodies (Sigma, St Louis, MO), and anti-keratin 12 (all diluted 1:300) were used to study cytokeratin expression [14]. Monoclonal antibody against a-smooth muscle actin (Sigma) was used to examine the possibility of transdifferentiation of keratocytes to myofibroblasts. Binding of the primary antibody was visualized with biotinylated secondary antibodies and substrate, according to the manufacturer's specifications (Vector Laboratories, Burlingame, CA). Skin samples of the wild type mice underwent the same immunohistochemical procedure and served as controls to validate the assays.

Northern hybridization

Total RNA was extracted from mouse tissues using TRI-reagent^TM (Molecular Research Center, Cincinnati, OH) as previously described [15]. Total RNA (10 mg) was subjected to electrophoreses in 1.3% agarose containing 2 M formaldehyde buffered with TBE (Tris-borate/EDTA). The RNAs were then transferred to Magna-Charge^TM membranes and hybridized with ³²P-labeled mouse keratocan, lumican, keratin 12, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probes in a hybridization solution containing 50% formamide at 41 °C overnight. The excess ³²P-probes were removed by high stringent washes with 0.1X SSC (1X SSC containing 0.15 M sodium chloride and 15 mM sodium citrate buffer, pH 7.0) and 1% SDS at 65 °C for 30 min each, repeated 3 times. Hybridization signals were detected with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA) as previously described [15,17]. The levels of keratocan, lumican, and keratin 12 mRNAs were compared to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in individual specimens.

Western blot analysis

Mouse corneas and skins were dissected and snap frozen in liquid nitrogen and pulverized with a mortar and pestle. Total proteins from one cornea or a skin sample sized 0.5 cm x 0.5 cm were extracted from the pulverized tissue by homogenization in a 20 mM Tris-HCl buffer, pH 7.4, containing 5 mM N-ethylmaleimide, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 5 mg/ml pepstatin (Sigma) at 4 °C. The resultant suspensions were centrifuged at 10,000 x g for 15 min at 4 °C. The supernatant was regarded as the soluble protein fraction and the pellets were further extracted with 20 mM Tris-HCl buffer, pH 7.4, containing 9 M urea, 2% SDS, 5% mercaptoethanol, 5 mM EDTA, 5 mM N-ethylmaleimide, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 5 mg/ml pepstatin at 4 °C. The extracts were centrifuged at 10,000 x g for 15 min at room temperature. The resultant supernatants were used for comparison with soluble proteins in a 5% SDS-polyacrylamide gel electrophoresis as previously described [4]. Proteins from unstained polyacrylamide gels were transferred electrophoretically onto Immobilon-P membranes (Millipore, Bedford, MA) using a semidry blotter apparatus. The membrane was incubated with anti-involucrin anitbodies (1:1000). Binding of the primary antibody was visualized using biotinylated secondary antibodies and substrate kits according to the manufacturer's specifications (Vector Laboratories).

In situ hybridization

To identify the cell types that express lumican, keratocan, and keratin 12, mouse eyes were fixed with 4% paraformaldehyde at 4 °C and embedded in paraffin as described previously [15,17]. Antisense and sense digoxigenin-labeled riboprobes (Boehringer Mannheim, Indianapolis, IN) of keratocan, lumican, and K12 mRNAs were synthesized and used for in situ hybridization on paraffin sections (5 mm) mounted on Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA) as previously described [15,17]. To remove nonspecifically bound probes, slides were subjected to a stringent wash with 0.5X SSC at 65 °C and treated with 20 mg/ml RNase (Sigma) at room temperature for 1 h, followed by washing with 0.2X SSC at 65 °C as described previously [15,17]. The hybridization signals were visualized with anti-digoxigenin antibody-alkaline phosphatase conjugates using procedures recommended by the manufacturer (Boehringer Mannheim).

Results

Immunochemical studies using keratinocyte differentiation markers

Homozygous corn1/corn1 mice at the age of 4 weeks demonstrate focal hyperplasia of corneal epithelium alternating with areas that appear to be normal, similar to observations previously reported (Figure 1) [13]. The keratin 12 expression pattern in the normal area of corn1/corn1 epithelium is similar to that of a wild type mice (Figure 1A and Figure 1C). Figure 1B and Figure 1E show the roughened corneal epithelial cells are present in the superficial layer of hyperplastic epithelium. In hyperplastic areas, both keratin 12 and keratin 14 are expressed most prominently in the basal and suprabasal layer of the corneal epithelium. In contrast, the expression of keratin 14 is restricted to the basal epithelial cells of wild type (Figure 1F) and in normal areas of the corneal epithelium of corn1/corn1 mice (Figure 1D and Figure 1F). In both wild type and corn1/corn1 mice, keratin 14 was diffusely detected in all epithelial layers at 7 and 14 days of age before the eye opens. Corneas of corn1/corn1 mice demonstrated many more desquamated cells in the space between cornea and eyelids, and about half of these desquamated cells stained with keratin 14 (unpublished observations). Anti-keratin 1 and anti-keratin 4 antibodies did not label corneal epithelial cells of wild type and corn1/corn1 mice (Figure 1G-L).

In normal areas of the corneal epithelium from corn1/corn1 mice and in corneas from wild type mice, involucrin was detected in most of the basal cells (Figure 2A and Figure 2C). However, in hyperplastic areas, immunostaining of involucrin was prominent in the basal and suprabasal layers of the corneal epithelium (Figure 2B). Loricrin and fillagrin antibodies did not stain corneas of either corn1/corn1 or wild type mice (Figure 2D-I). Using the same procedure, loricrin, involucrin, fillagrin, and keratin 1 were detected in the superficial epidermal layers of skin, the tissue location of these proteins (data not shown).

To further confirm the presence of involucrin in the cornea, proteins were extracted with Tris buffer (soluble fraction) and with a 9 M urea solution (insoluble fraction) from corneas and skin of wild type and corn1/corn1 mice. The samples were subjected to western blot analysis with anti-involucrin antibodies. As shown in Figure 3, two proteins of 98 kD and 110 kD were detected by the antibodies in the soluble fraction from both corneas and skin of wild type mice. In the soluble fraction prepared from corn1/corn1 mice, only the 98 kD protein was detected. None of the proteins were found in the urea-extracted fraction from corneas of both corn1/corn1 and wild type mice, while both were detected in skin of corn1/corn1 and wild type mice. The nature of these two polypeptides with which the antibodies react is unknown. However, a variation of post-translational modifications, e.g., glycosylation, phosphorylation, and/or proteolytic reaction, may account for the different mobility in gel electrophoresis.

Northern and in situ hybridizations of stromal cell markers

During corneal wound healing, neovascularization is often accompanied by changes in keratocyte characteristics, e.g., synthesis of specific extracellular components such as: lumican, keratocan, and an aberrant expression of a-smooth muscle actin signifying the phenotype of myofibroblasts. To examine whether keratocyte differentiation might be involved in corn1/corn1 mice, the expression of keratocan, lumican, and keratin 12 mRNAs were examined by northern and in situ hybridization (Figure 4). The levels of lumican and keratocan mRNA in corneas of corn1/corn1 mice were higher than that of wild type mice (Figure 4A). In wild type mice, the expressions of lumican and keratocan mRNAs were restricted to keratocytes (Figure 4B). In corn1/corn1 mice, lumican mRNA was also detected in epithelial cells in addition to keratocytes, while the keratocan mRNA was still only detected in keratocytes.

To investigate the possibility of a transformation of keratocytes into myofibroblasts in corn1/corn1 mice, expression of a-smooth muscle actin (a-SMA) was assessed in keratocytes of corn1/corn1 mice by immunohistochemistry. The anti-a-SMA antibody did not label stromal cells of corn1/corn1 and wild type mice, but did label the smooth muscle cells of blood vessels in the mutant mice. This observation suggests that stromal keratocytes of corn1/corn1mice do not undergo transdifferentiation to myofibroblasts, in the age group investigated.

Discussion

In the present studies, we examined the expression patterns of keratins by corneal epithelial cells, and lumican and keratocan by stromal keratocytes in corn1/corn1 mice. Our results indicate that epithelial hyperplasia of corn1/corn1 mice is characterized by changes in the expression patterns of keratin14 and involucrin and lumican by corneal epithelial cells, but the expression of keratocan is still limited to keratocytes.

Phenotype of corneal epithelial cells

Expression of cytokeratins is tissue-specific and differentiation-dependent [1-4]. Imunohistochemistry with specific antibodies against keratins has been widely used as one of the criteria for the identification of cell lineages of carcinoma and of other abnormal tissues [1,2]. Keratin 14 is a differentiation marker of basal keratinocytes, but can be detected in suprabasal cells of hyperplastic epithelium in certain disease processes, e.g., hyperplasia or dysplasia of oral mucosa and skin [18-20]. Likewise, the expression of keratin 14 by both basal and suprabasal layers of hyperplastic epithelium indicates hyper proliferative activities of corneal epithelial cells of corn1/corn1 mice. In contrast, the expression of keratin 12 is expressed throughout all corneal epithelial cell layers of both wild type and corn1/corn1 mice. Anti-keratin 1 and anti-keratin 4 antibodies did not label the corneal epithelium of corn1/corn1 mice. These observations indicate that epithelial hyperplasia does not cause transdifferentiation of corneal epithelium, since they do not express keratin 1 and keratin 4, markers of epidermal and conjunctival epithelium, respectively.

The immunohistochemical studies revealed that basal corneal epithelial cells of wild type mice express involucrin, which was also detected in both basal and suprabasal corneal epithelial cells of corn1/corn1 mice. It has been suggested that involucrin serves as an initiator for the formation of the cornified envelope that is a characteristic of epidermis [21]. Thus, the expression of involucrin signifies the beginning of keratinocyte terminal differentiation. The cornified envelope is a highly insoluble structure assembled close to the plasma membrane of stratified squamous epithelium that is essential for effective barrier function [1,21-25]. Cornification of epithelium involves the expression of a group of specific proteins, such as cystatin a, keratolinin, elafin, involucrin, loricrin, and members of the small proline-rich superfamily (Spr), fillagrin, keratin intermediate filaments, and trichohyalin [25]. These components become insoluble and crosslinked via disulfide and isopeptide bonds by disulfide oxidase and transglutamylase, respectively [26]. However, in the present study, we used western analysis to demonstrate that involucrin could exist as a soluble protein in corneas of wild type and corn1/corn1 mice (Figure 3). The significance of only a 98 kD protein present in the cornea of corn1/corn1 mice remains elusive. However, our observations are consistent with the notion that involucrin molecules are likely not crosslinked by covalent bonds in the corneal epithelium of wild type and corn1/corn1 mice. This suggestion is further supported by experiments in which immunohistochemistry also failed to detect the presence of other components of cornified envelope, i.e., keratin 1, loricrin, and profillagrin/fillagrin in corneal epithelium (Figure 2). Thus, the existence of involucrin does not necessarily lead to cornification of corneal epithelium. An implication of the finding is that involucrin may have functions other than serving as a component of the cornified envelope in the stratified corneal epithelium. However, the function of involucrin in corneal epithelial cells remains unknown.

Altered keratin 14 expression has been observed in many corneal diseases involving epithelia hyperplasia, suggesting that pathogenesis tends to induce high proliferation in epithelial cells [18]. The mechanism of increased expression of keratin 14 in the hyperplastic regions of corneal epithelium observed in corn1/corn1 mice remains unknown. The factor(s) involved in corneal neovascularization and epithelial hyperplasia do not disrupt corneal type epithelial differentiation of keratin 12 expression. This observation differs from that of our previous studies in which we demonstrated that an overexpression of FGF-7 under the control of lens cell-specific aA-crystallin promoter could alter the fate of the ocular surface ectoderm and lead to the replacement of the corneal surface with conjunctival epithelium in transgenic mice [12]. The differences in the phenotypes in FGF-7 overexpressing transgenic and corn1/corn1 mice may be in part explained by the variation of temporal expression of excess growth factors during eye development. In FGF-7 overexpressing transgenic mice, these growth factors were expressed early during development prior to the commitment of the corneal epithelium to a corneal type of epithelial differentiation. This is supported by the observation that in corn1/corn1 mice the corneal epithelium appears normal during embryonic development [13]. Neovascularization does not take place until the eyes open at postnatal day 14 at a time the corneal epithelium has already stratified. Therefore, our findings are consistent with the notion that corneal epithelial cells do not undergo transdifferentiation under the influence of factors associated with Corn phenotype during embryonic development of corn1/corn1 mice.

Phenotype of stromal cells

Keratocan and lumican are the two major keratan sulfate proteoglycans found in the corneal stroma. Both keratocan and lumican are downregulated during the wound healing [27]. In contrast, the neovascularization in the corneal stroma of corn1/corn1mice was accompanied with up regulation of keratocan and lumican mRNAs. During wound healing, keratocytes undergo phenotypic changes and express a-smooth muscle actin, a characteristic of myofibroblasts in granulation tissues [28]. The absence of a-smooth muscle actin in the stromal keratocytes of corn1/corn1 mice indicates that the keratocytes did not transdifferentiate to myofibroblasts in corn1/corn1 mice, at the age period studied. The up-regulation of keratocan and lumican in corn1/corn1 mice may result from factors associated with neovascularization or altered epithelial cell functions.

Keratocan mRNA is selectively expressed by the corneal keratocytes of the adult mouse [15,17]. In contrast, lumican is expressed by corneal epithelium during development, by the corneal keratocytes, and many other tissues of adult mice, e.g., tendon, heart, dermis, etc [15]. Injured mouse corneal epithelium transiently expresses lumican during the early phase of wound healing [29]. Thus, we suggest that corneal epithelial hyperplasia may express lumican, whereas the expression pattern of keratocan remained restricted to corneal stromal keratocytes of corn1/corn1 mice.

The results of present studies support the notion that ocular surface epithelial cells, once committed to become corneal epithelial cells, do not undergo transdifferentiation under the influence of growth factors and cytokines accompanying inflammation and/or neovascularization.

Acknowledgements

Supported in part by grants from NIH Core Center Grant CA 34196, EY 10556, EY 11837 and Ohio Lions Eye Research Fundation.

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