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Human Molecular Genetics, 2003, Vol. 12, No. 9 1029-1036
DOI: 10.1093/hmg/ddg112
© 2003 Oxford University Press

Aberrant actin cytoskeleton leads to accelerated proliferation of corneal epithelial cells in mice deficient for destrin (actin depolymerizing factor)

Sakae Ikeda, Leslie A. Cunningham, Dawnalyn Boggess, Craig D. Hobson, John P. Sundberg, Jürgen K. Naggert, Richard S. Smith* and Patsy M. Nishina

The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA

Received December 20, 2002; Accepted February 24, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Corneal disease is the most common cause of bilateral blindness in the world. Visual loss in this condition is often due to changes in morphology and function of the corneal epithelial surface. Corneal disease-1 (corn1) and corn12J are spontaneous mouse mutants that develop irregular thickening of the corneal epithelium, similar to that observed in human corneal surface disease. These autosomal-recessive mutations cause an increase in the rate of proliferation of the corneal epithelial cells. Here, we report that the phenotypes in both mutants are caused by mutations within the destrin gene (also known as actin-depolymerizing factor). By positional cloning, we identified a deletion encompassing the entire coding sequence of the destrin gene in corn1 mice, and a point mutation (Pro106Ser) in the coding sequence of destrin in corn12J mice. In situ analysis showed that destrin is highly expressed in the corneal epithelium. Consistent with the cellular roles for destrin, an essential regulator of actin filament turnover that acts by severing and enhancing depolymerization of actin filament, we observed that the corn1 mutations increased the content of filamentous actin in corneal epithelial cells. Our results suggest an in vivo connection between remodeling of the actin cytoskeleton and the control of cell proliferation, and a new pathway through which an aberrant actin cytoskeleton can cause epithelial hyperproliferation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The cornea is located at the most anterior region of the eye and its smooth surface and transparency are necessary for the proper refraction of light. Corneal disease is responsible for 6 % of legal blindness in the USA and is the most common cause of bilateral blindness in the world (1). Visual loss in many corneal diseases is due to changes in morphology and function of the corneal epithelial surface. Irregular thickening of the corneal epithelium can cause corneal scarring, increase susceptibility to infection, and is frequently associated with corneal stromal neovascularization (2–5). Ocular disorders that cause surface irregularity of the cornea include keratitis sicca, xerophthalmia, chemical and thermal burns, ocular cicatricial pemphigoid, Stevens–Johnson syndrome, and numerous dermatological diseases (6). To determine the etiology of these human conditions, it is important to understand the mechanisms underlying the normal maintenance of the corneal epithelial integrity and function, and the molecular pathways through which irregular thickening of this tissue is caused.

Mice with a spontaneous autosomal recessive mutation, corneal disease-1 (corn1), develop a roughened opaque corneal surface accompanied by corneal stromal neovascularization (7). Irregular thickening of the corneal epithelium is evident by 1 week of age and neovascularization in the corneal stroma is observed by 18 days of age. Cell kinetic studies have demonstrated that thickening of the corneal epithelium in corn1 mice is caused by a dramatic increase in the rate of proliferation of the corneal epithelium (7). The corn1 mouse offers a unique model to evaluate the regulatory factors that control corneal epithelial proliferation and the processes at work in human corneal surface diseases. Here, we report that the gene mutated in the corn1 and in the newly identified corn12J strains encodes destrin (also more commonly known as actin-depolymerizing factor or ADF). Destrin is an essential actin regulatory protein belonging to the ADF/cofilin family (8,9), that binds to actin subunits in filamentous actin (F-actin) (10), enhancing the subunit off-rate (11) and promoting filament severing (12). This family of proteins is responsible for enhancing the turnover of actin in vivo. These results demonstrate that the proper regulation of actin dynamics is necessary for normal maintenance of the corneal epithelium and suggest a new pathway through which aberrant actin cytoskeleton leads to epithelial proliferation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of corn1 as a deletion of the destrin gene
The corn1 mutation had been previously mapped to mouse chromosome 2 (7). A high-resolution genetic map of the corn1 locus was generated using intra-subspecific F2 intercrosses between A.BY-H2bH2-T18b/SnJ (A.BY)-corn1/corn1 and strains CAST/EiJ, DBA/2J or LP/J. The critical interval of 0.13±0.06 cM, defined by the intercrosses, contained five recombination events between the flanking markers, D2Mit513 and D2Mit491. Since additional markers were necessary to narrow the interval further, a human and mouse BAC physical map of the critical region was assembled, utilizing the sequence homology between the corn1 interval on mouse chromosome 2 and human chromosome 20p11.2 (Fig. 1). By genotyping recombinant mice with polymorphic markers generated using P1 and BAC sequences, the corn1 region was further narrowed to 0.11±0.05 cM flanked by markers D2Mit513 and D2Pjn420-9. Five transcripts were present in the annotated human BAC sequences that covered the corn1 minimal region and were tested for alterations in coding sequence and expression level between corn1 and wild-type mice. The results indicated that a mutation within the destrin (Dstn) gene was responsible for the corn1 phenotypes. Northern analysis demonstrated absence of Dstn mRNA expression in homozygous corn1 mice (Fig. 2A). PCR analyses of genomic sequences (Celera database) established the presence of a ~35 kb deletion that encompasses the entire coding sequence of Dstn in corn1 mice (Fig. 2B).



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  		Figure 1. A physical map of the corn1 region. Polymorphic markers, D2Mit513, D2Mit491 and D2Mit194 were initially used to screen the mouse P1 library. Blast searches with mouse sequences yielded human BAC clones from 20p.12 and a contig of eight overlapping BACs that covered the corn1 region. A mouse BAC clone (RP23-420F12) was also identified by blast searches using human sequences. Dotted lines from the mouse sequences to human sequences indicate that homology was observed between them. Additional polymorphic markers were designed to narrow the corn1 minimal region, which was finally flanked by D2Mit513 and D2Pjn420-9 (shown in rectangular boxes). PCSK2, proprotein convertase subtilisin/kexin type 2; BFSP1, beaded filament structural protein 1; RRBP1, ribosome binding protein 1; SNX5, sorting nexin 5.

 


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  		Figure 2. Identification of corn1 and corn12J mutations. (A) The corn1 mutation leads to the absence of Dstn mRNA expression. Northern analyses of eye polyA RNA (5 µg) from corn1 and A.BY-wild type mice are shown. The membrane was sequentially hybridized with the Dstn probe (top panel) and the Gapd probe (bottom panel). (B) A schematic figure showing a ~35 kb genomic deletion in the corn1 mouse. A primer set F29–R13 was used to amplify a 600 bp fragment in corn1 mice. Nucleotide sequences at junctions of the deletion are shown. Nucleotides in blue are only present in the corn1 mice. (C) corn12J mice (right panel) show a mild corneal epithelial phenotype. The epithelial lesion is characterized by focal epithelial thickening and enlarged surface epithelial cells with degenerating nuclei (arrow). (D) Electropherograms showing a point mutation in the Dstn gene in corn12J mice. The mutation causes a proline->serine amino acid change.

 
Identification of the corn12J mutation
During positional cloning of the corn1 locus, an additional spontaneous, independent allelic mutation of corn1 (named corn12J) was discovered in the C57BL/6JSmn (B6Smn) inbred strain. Allelism was confirmed by complementation tests with A.BY-corn1/corn1 mice. Unlike corn1 mice, corn12J mice completely lack neovascularization and have a milder corneal epithelial phenotype (Fig. 2C). Mutational analysis of the Dstn gene in corn12J mice demonstrated a C->T transversion at nucleotide position 316 in exon 3 that causes a Pro106Ser amino acid change (Fig. 2D). Identification of the corn12J mutation further confirms that Dstn is indeed the gene that is responsible for corn1 phenotypes. The 106Pro residue is conserved across species (8,13) and is located in the loop connecting the long {alpha}-helix (referred to as {alpha}5 in 14) and the adjacent ß sheet (ß3 in 14). Analyses of the three-dimensional structure of yeast cofilin indicate that residues that have been implicated in both F- and G-actin binding by the mutagenesis study, cluster around this region (15). This suggests that the corn12J mutation might affect the binding of destrin to F- and G-actin.

Expression of destrin in the cornea
In order to evaluate the expression of destrin in the cornea, we performed in situ hybridization and immunohistochemical studies. In wild-type mice, Dstn mRNA was highly expressed in the corneal epithelium, but not in the corneal stroma or endothelium (Fig. 3). In contrast, Dstn expression was absent in the corneal epithelium of corn1 mice (Fig. 4C), consistent with the results of our northern analysis. Immunohistochemical analysis using an anti-chicken ADF antibody (generous gift from Dr James Bamburg) that recognizes DSTN and cofilins in the mouse (16) showed cytoplasmic immunoreactivity of corneal epithelial cells (Fig. 4A). The fainter signal detected in corn1 corneal sections (Fig. 4B), where DSTN should be absent, probably represents cofilin-1. This result suggests that DSTN is the major ADF/cofilin molecule expressed in the cornea. This hypothesis is supported by immunoblotting results which confirm a high abundance of DSTN in wild-type cornea (Fig. 4C). Interestingly, cofilin-1 levels in the cornea of corn1 mice were significantly increased over wild-type controls.



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  		Figure 3. Expression of Dstn mRNA in the cornea. Bright-field (A, C) and corresponding dark-field sections (B, D) are shown. (A, B) In wild-type mice, Dstn mRNA is highly expressed in the epithelium (Ep) of the cornea. (C, D) In corn1 mice, no signal was detected. Ep, corneal epithelium; S, corneal stroma; En, corneal endothelium.

 


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  		Figure 4. Expression of DSTN and cofilins in the corneal epithelium. (A) In wild-type mice, strong DSTN/cofilins signal is detected in the cytoplasm of the corneal epithelial cells. (B) In corn1 mice, only a faint signal is detectable representing cofilin-1 expression. (C) Western blots probed with the antibody recognizing DSTN and cofilins (top panel) and the antibody specific to cofilins (bottom panel). Extracts of the wild-type (+/+) and corn1/corn1 cornea and recombinant chicken ADF (positive control) were subjected to SDS–PAGE. In the wild-type cornea, a strong DSTN/cofilins signal was detected. In the corn1 cornea, a fainter band was detected at a slightly higher molecular weight, which represents cofilin-1. Note that the cofilin signal in corn1 mice is much stronger compared with that in +/+ mice.

 
Corn1 mutations increase F-actin content in corneal epithelial cells
To evaluate how mutations in destrin affect its substrate, F-actin, in corneal epithelial cells, corneal whole-mounts were stained with FITC-phalloidin. In wild-type mice, F-actin staining was primarily observed in the cortex of corneal epithelial cells (Fig. 5A and C). However, in both corn1 and corn12J mice, the corneal epithelial cells demonstrated a marked increase in F-actin levels in a pattern similar to stress fibers (Fig. 5B and D). These results demonstrate that the corn1 mutations indeed affect the organization of the actin cytoskeleton in corneal epithelial cells.



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  		Figure 5. Whole-mount F-actin staining of the wild-type and corn1 cornea. In the cells of the corneal epithelial surface, while A.BY wild-type (A) and B6 wild-type (C, C') mice exhibit weak cortical expression, A.BY- corn1/corn1 (B) and B6Sm- corn12J/corn12J (D) mice show a markedly increased level of expression in a pattern similar to stress fibers. Images in (A)–(D) were obtained using identical settings; (C') is an image of the same view as (C) taken at increased contrast and brightness to show the cortical signal observed in wild-type mice. Scale bar: 20 µm.

 
Neovascularization in corn1 but not corn12J is due either to allelic differences or a closely linked locus
In order to examine whether phenotypic differences between corn12J and corn1 mice are due to allelic differences or genetic background effects, we carried out a backcross, [F1(B6Sm-corn12J/corn12JXA.BYSn/J-corn1/corn1)]xA.BYSn/J-corn1/corn1. In 44 backcross offspring examined by slitlamp biomicroscopy, epithelial proliferation and neovascularization cosegregated. That is, all offspring that showed a severe epithelial phenotype developed neovascularization and offspring that showed a mild epithelial phenotype did not. Mice that carried two corn1 alleles showed both epithelial proliferation and neovascularization, while mice with one corn1 allele and one corn12J allele showed only epithelial proliferation. Chi-square analysis indicated this difference to be significant ({chi}2=40.16, P<10-9). In addition, in a genome-wide linkage analysis of the backcross, no unlinked loci were detected that modified epithelial proliferation or neovascularization (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Identification of the molecular basis of human ocular surface diseases and animal models of these conditions has provided valuable information regarding the pathways that are critical for normal function and maintenance of the corneal epithelium. For example, mutations within cornea-specific keratins, K3 and K12, which lead to Meesmann's corneal dystrophy, indicate that these intermediate filaments are essential for the maintenance of corneal epithelial integrity and structural alteration of these proteins cause fragility of the corneal epithelium (17–20). Another gene that has been associated with aberrant corneal epithelialization is a gastrointestinal tumor-associated antigen, M1S1. Mutations in M1S1cause gelatinous drop-like corneal dystrophy (GDLD) characterized by severe corneal amyloidosis (21–24). Since this molecule had been suggested to function as a cell–cell adhesion molecule (25), this led investigators to examine corneal epithelial ultrastructure and epithelial barrier function in GDLD patients (26). The results from this study suggested that mutations in M1S1 may lead to abnormal corneal epithelial cell junctions which cause increased cell permeability, which in turn may contribute to the pathogenesis of amyloid deposition. As in previous studies, the identification of the molecular basis of corn1 as destrin (Dstn) has lead to insights into the pathways through which proliferation of corneal epithelial cells is controlled.

The corneal epithelium is a self-renewing tissue whose stem cells reside in the most peripheral regions of the cornea called the limbus (27–29). Corneal stem cells undergo occasional cell division, giving rise to rapidly dividing transiently amplifying cells. Transiently amplifying cells are located at the basal layer of the corneal epithelium and undergo several rounds of cell division (28) as they migrate centrepetally (30,31). In the corn1 mouse, this process is disrupted and accelerated, and uncontrolled proliferation of basal corneal epithelial cells is observed. Consistent with the cellular role destrin plays to enhance actin dynamics by depolymerizing and severing filamentous actin, accumulation of F-actin was observed in corneal epithelial cells of corn1 mice. This result suggests that actin filament turnover mediated by destrin is critical for proper cell cycle progression of corneal epithelial cells. This notion is also supported by in vitro studies that have shown a requirement for intact actin dynamics in the regulation of cell cycle progression. In these studies, various drugs known to inhibit different steps in actin assembly/disassembly also inhibited cell cycle progression in cultured cells (32–36). Cytochalasin D and latrunculin B reduce actin turnover by inhibiting actin polymerization (37–40), while jasplakinolide inhibits actin depolymerization thereby reducing turnover rate (41). The cell-cycle arrest by cytochalasin D was also accompanied by an upregulation of the cell-cycle inhibitor, p27Kip1 (36). Although these drugs cause cell cycle arrest rather than the hyperproliferation observed in corn1 mice, our findings provide an in vivo connection between remodeling of the actin cytoskeleton and the control of cell cycle progression and proliferation in corneal epithelial cells.

Remodeling of the actin cytoskeleton through the action of ADF/cofilins is fundamental for many basic processes including cell polarization, cell motility, cell division, endocytosis and exocytosis (8,42,43). Consistent with their basic cellular roles, studies in model systems such as yeast and Drosophila have shown that loss of ADF/cofilin function leads to lethality (44–46). In light of these facts, it is rather surprising that mutant phenotypes of corn1 mice appear to be restricted to the cornea (7). Our results suggest that in sites where DSTN and cofilin-1 are both normally expressed, cofilin-1 may, in part, compensate for the loss of DSTN. While destrin is distributed in many organs (47), its expression is restricted to epithelial and endothelial cells (14). Cofilin-1 is ubiquitously expressed in most cell types throughout development and adulthood, while cofilin-2 is a muscle specific isoform (14). In vitro studies have shown that, although DSTN is more efficient and shows stronger pH-dependent activity, both DSTN and cofilin-1 are able to turn over actin filaments (11,14). Our results suggest that their functions are indeed overlapping in vivo. The compensatory mechanism between DSTN and cofilin-1, however, is not sufficient in the cornea. In spite of the upregulation in cofilin-1 expression in the cornea of corn1 mice, it is unable to fully regulate actin filament dynamics and compensate for the loss of DSTN.

Availability of two corn1 alleles on different genetic backgrounds with different phenotypic expressions enabled us to carry out genetic analyses to investigate the basis for the phenotypic differences between A.BY-corn1 and B6Smn-corn12J. The milder phenotype observed in corn12J mice with smaller areas of focal proliferation and no neovascularization could either be due to allelic differences or genetic background effects (A.BY versus B6Smn). The results of our genetic analyses suggest that the difference in phenotypes observed in the two mutants is likely to be due to allelic differences between corn1 and corn12J rather than to a genetic background effect. Perhaps the corn12J allele is partially functional, and hence causes a milder phenotype, whereas the corn1 mutation which deletes the entire destrin coding region is a functional null allele. We cannot, however, rule out the possibility that a factor that is very closely linked to the corn1 locus may account for the phenotypic variation. Biochemical analyses of destrin function in both mouse models should help to clarify this issue. It also remains to be examined whether corneal neovascularization observed in corn1 but not in corn12J mice is due to actin cytoskeletal defects in vascular cells or a paracrine effect of the hyperproliferative epithelium. Further genetic and molecular analyses using corn1 and corn12J mice will help to elucidate the mechanism controlling epithelial and blood vessel proliferation in the cornea.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Genetic crosses and mapping
A.BY-corn1/corn1 mice were crossed to CAST/EiJ, DBA/2J and LP/J and progeny were intercrossed to produce 1862 F2 mice. Phenotyping was performed by slit-lamp microscopy (7). Genotyping with Chromosome 2 SSLP markers that are polymorphic between strains A.BY and CAST/EiJ, A.BY and DBA/2J or A.BY and LP/J was carried out as previously described (48). Mice recombinant between flanking markers were progeny tested by crossing to A.BY-corn1/corn1 to determine whether these mice carried the corn1 locus.

Mouse P1/BAC and human BAC screening
We identified mouse P1 clones by PCR-based screening of a 129P2/Ola P1 library with DNA markers, D2Mit513, D2Mit194 and D2Mit491, and markers generated from end sequences of P1s. Human BAC sequences were obtained from the NCBI database by blast searches with mouse sequences. A mouse BAC clone (RP23-420F12) was identified by blast searches using human sequences.

Northern analysis
Northern hybridization was performed as previously described (49). A membrane with 5 µg poly(A)+ RNA from wild-type and corn1 mouse eyes was hybridized with a [32P] random-labeled cDNA probe for ADF (nt 5–638, AB025406). A mouse Gapd probe was used as a control for equal RNA loading.

In situ hybridization
Four-week-old A.BY-corn1/corn1 and wild-type mice were anesthetized with tribromoethanol and perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Eyes were enucleated and post-fixed in the same fixative for 18 h, dehydrated and embedded in paraffin. Sections were hybridized with [{alpha}-33P]UTP-labeled sense and antisense riboprobes (105 cpm/µl) generated from a plasmid containing a cDNA fragment of destrin nt 5–638 (AB025406) as previously described (50).

Immunofluorescence
Four-week-old A.BY-corn1/corn1 and wild-type mice were euthanized by carbon dioxide (CO2) asphyxiation. Eyes were enucleated and placed in 4% PFA in PBS overnight, dehydrated and embedded in paraffin. Sections were incubated overnight with anti-ADF primary antibody (rabbit 1439) (16). The anti-ADF antibody produced against chicken ADF recognizes not only destrin but also cofilins in mouse (16). Destrin and cofilins which share similar function are ~70% identical (14). Antibody binding was detected using biotinylated secondary antibodies (Vector Laboraotories) followed by FITC-AvidinD (Vector Laboraotories). Sections were heated in the microwave for 8 min in sodium citrate buffer, pH 6.5 for antigen retrieval prior to incubation with the primary antibody. Images were collected on a Leica DMRXE fluorescent microscope equipped with a SPOT CCD camera (Diagnostic Instruments) using an appropriate bandpass filter.

Immunoblotting
Protein extracts from A.BY-corn1/corn1 and wild-type corneas (10 µg) were separated on 17% SDS–polyacrylamide gel and electrotransferred onto PVDF membranes (Roche). The membranes were blocked in Tris-buffered saline (TBS) containing 5% fat-free milk and incubated with primary antibodies: anti-ADF (rabbit 1439, equally reactive to rodent destrin and cofilins) (16) and anti-cofilin (Cytoskeleton). We incubated the membranes with a peroxidase-conjugated secondary antibody and detected signal using chemiluminescent reagents (Amersham).

Whole-mount F-actin staining
A.BY-corn1/corn1, B6Smn-corn12J/corn12J and wild-type control mice were euthanized by CO2 asphyxiation. Eyes were immediately fixed in situ with 4% PFA in PBS for 20 min, enucleated, placed in 4% PFA for additional 30 min and washed in PBS. Corneas were removed under a dissecting microscope, incubated with FITC-phalloidin (Sigma) for 2 h at room temperature and washed in PBS. The corneas were mounted in Slow Fade (Molecular Probes) with the epithelium side up on glass slides, covered with coverslips and viewed with a confocal laser scanning microscope. Whole-mounted corneas were viewed with a Leica TCS NT confocal laser scanning microscope equipped with an argon laser. The FITC-detector channel was used with the excitation wavelength of 488 nm and the emission barrier filter at 515 nm.


   ACKNOWLEDGEMENTS
 
We thank Drs Simon W.M. John, Timothy P. O'Brien and Barbara K. Knowles for careful review of the manuscript. We are also grateful to Dr James R. Bamburg for providing us with the ADF antibody and for reviewing the manuscript. This work was supported by grants from the National Eye Institute. Institutional shared services are supported by a National Cancer Institute Support grant.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 2072886283; Fax: +1 2072886078; Email: rss{at}jax.org Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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