[Show abstract][Hide abstract] ABSTRACT: Immortalized cell lines representing fibroblast cells from corneal stroma would facilitate studies of corneal cell biology and injury response.
Primary cultures of cells derived from mouse corneal stroma were transfected with a human telomerase reverse transcriptase (hTERT) expression construct to maximize chances of cellular immortalization. A resulting cell line was analyzed for telomerase activity, cell growth characteristics, senescence and gene expression patterns. Specific responses to transforming growth factor beta (TGF-beta) were also analyzed.
An immortalized cell line was derived and was named MK/T-1. MK/T-1 cells show no signs of cellular senescence or transformation at over 100 passages. Telomerase activity was significantly higher in MK/T-1 cells as compared to the parental cell cultures. However, relative telomere length (RTL) in the MK/T-1 and parental cells was not significantly different. Senescence associated beta-galactosidase (SA-beta-Gal) activity was not detected in late passage MK/T-1 cells while the parental cells had already upregulated SA-beta-Gal at high levels by passage 9. The MK/T-1 cells express vimentin, tubulin, lumican, mimecan, decorin and collagen I, but not keratocan. Exposure of the MK/T-1 cells to TGF-beta induces the expression of smooth muscle alpha-actin (ASMA), the activation of MAP Kinase (p38-MAPK) and morphological changes consistent with cytoskeletal reorganization.
MK/T-1 cells represent an immortalized fibroblast cell line derived using cultures from corneal stroma cell preparations. Expression of hTERT may contribute to immortalization of the MK/T-1 cells by a mechanism other than increases in RTL. MK/T-1 cells may be a useful model in which to study the responses of corneal fibroblast cells to cytokines and other diverse environmental factors in vitro.
[Show abstract][Hide abstract] ABSTRACT: The human lumican gene was shown to possess one major transcription start site, resulting in exon 1 of the gene giving rise to the first 74 base pairs (bp) of the 5'-untranslated region. About 1.6 kilobase pairs of upstream promoter sequence were sequenced and analyzed to identify elements responsible for gene expression. No typical TATAA sequence was identified in the vacinity of the transcription start site, but an atypical TATCA sequence residing 41 bp upstream was shown to be necessary for transcription, although it was incapable of supporting transcription by itself. A GC box residing 74 bp upstream of the transcription start site also was essential for the initiation of transcription. Sp3 was identified as the transcriptional activator binding to the GC box. No additional elements that significantly modulated transcription were noted in the promoter sequence analyzed, when using human adult chondrocytes as the cell source for transfection in reporter assays. In contrast, reporter assays carried out in human fetal lung fibroblasts, where lumican expression is deplete, revealed the presence of a repressor element located between 384 and 598 bp upstream of the transcription start site. A GATA-binding site located between bp -386 and -391 was identified as being necessary for repression of transcription. The mouse lumican promoter does not possess an equivalent site, and this may explain why the lumican gene is expressed in fetal murine cartilage but not in fetal human cartilage.
[Show abstract][Hide abstract] ABSTRACT: Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e. g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum(-/-) mice was significantly delayed compared with Lum(+/-) mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.
[Show abstract][Hide abstract] ABSTRACT: Keratin 12 (K12) is a cornea epithelial cell-specific intermediate filament component. To provide a better understanding of its expression, it is necessary to identify and characterize the promoter of Krt1.12 gene.
The 2.5-kb DNA 5' to Krt1.12 gene was sequenced. Krt1.12 promoter-beta-gal DNA constructs were prepared and used in vivo to transfect rabbit corneas, conjunctivas, and skin by particle-mediated gene transfer (Gene Gun). In vitro, the DNA constructs were transfected into cultured T-antigen-transformed rabbit corneal epithelial (RCE-T) cells and human fibrosarcoma HT-1080 fibroblasts with lipofectamine. The promoter activity was assessed by measuring beta-gal (beta-galactosidase) activity using histochemical staining with 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside and enzyme assay with o-nitrophenyl beta-D-galactopyranoside.
There are four Pax-6 pair box binding elements found between -910 and -2000 bp 5'-flanking the transcription initiation site of the Krt1.12 gene. None of promoter constricts can be expressed by HT-1080 cells. Cotransfection of Pax-6 cDNA with K12 promoter-beta-gal constructs containing Pax-6 elements results in a fourfold increase of beta-gal activities in RCE-T cells but not HT-1080 fibroblasts. The data of in vivo transfection in the rabbit by Gene Gun indicate that reporter gene constructs containing 0.6-kb and longer DNA fragments 5'-flanking Krt1.12 gene are effectively expressed in corneal, but not conjunctival or epidermal epithelial cells.
The particle-mediated gene transfer is a suitable technique for in vivo delivery of transgenes to corneal epithelial cells. The 2.5-kb DNA fragment 5'-flanking Krt1.12 contains corneal epithelial cell-specific regulatory cis-DNA elements. Pax-6 is a positive transcription factor essential for keratin 12 expression.
[Show abstract][Hide abstract] ABSTRACT: Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentiation. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques.
One allele of murine Krt1.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization.
The heterozygous mice (+/-) one allele of the Krt1.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (-/-) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Northern hybridization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of -/- mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in -/- mice decreases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial, cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of -/- mice.
The presence of cornea-specific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.
[Show abstract][Hide abstract] ABSTRACT: Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization.
Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross.
Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology.
This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.
[Show abstract][Hide abstract] ABSTRACT: Keratins are a group of water-insoluble proteins constituting paired acidic and basic keratin molecules that form 10-nm intermediate filaments in epithelial cells. Expression of the K3/K12 keratin pair characterizes the cornea-type differentiation. However, the mechanism that regulates this cornea-specific K12 expression remains unknown. To provide a better understanding of the cornea-specific expression, we have cloned the K12 cDNA (Liu, C.-Y., Zhu, G., Westerhausen-Larson, A., Converse, R., Kao, C. W.-C., Sun, T.-T., and Kao, W. W.-Y. (1993) Curr. Eye Res. 12, 963-974). In present studies, the murine K12 keratin gene (Krt1.12) was isolated and characterized. The murine Krt1.12 gene spans 6,567 base pairs of genomic DNA, and the mRNA encoding K12 keratin is distributed into eight exons. Chromosome mapping reveals that murine Krt1.12 is located within the Krt1 complex of mouse chromosome 11. In addition to the production of authentic K12 mRNA, the Krt1.12 gene gives rise to several alternate poly(A)+ RNAs by the use of alternative splicing in intron 2, an alternative promoter in intron 1, and/or both. Sequence analysis indicates that the transcripts derived from alternative splicing and/or the alternative promoter do not have a long open reading frame for keratin or keratin-like molecules. It is not known whether these alternate K12 poly(A)+ RNAs have any biological functions, e.g. regulation of K12 gene expression.
Journal of Biological Chemistry 11/1994; 269(40):24627-36. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The conjunctival epithelium is intrinsically different from the corneal epithelium in vivo, but sometimes can transform into an epithelium morphologically indistinguishable from the latter after healing of a total corneal epithelial defect. It remains unclear whether this morphologic transformation represents a process of extrinsic modultation or transdifferentiation of intrinsically divergent epithelium. In air-lifted organotypic cultures, rabbit conjunctival epithelial cells lost goblet cell differentiation and were stratified to the same extent as corneal epithelial cells, resembling the above in vivo morphologic transformation. Paired expression of K3 (64 kD) and K12 (55 kD) keratins has been regarded as a marker for corneal-type differentiation. Immunoblot analysis by monoclonal antibody AE5 revealed that K3 keratin was expressed by both submerged or air-lifted corneal and conjunctival cultures with or without 3T3 fibroblasts in collagen gel. In contrast, K12 keratin was expressed only by air-lifted corneal cultures with 3T3 fibroblasts using monoclonal antibody AK2 and two epitope-specific antibodies to N- and C- terminal oligopeptides deduced from the mouse K12 gene. This finding was also confirmed by Northern hybridization with a rabbit K12 cDNA probe. The expression of K12 keratin was more delayed than that of K3 keratin in air-lifted corneal cultures. This dissociated expression of these two keratins resembles that noted in vivo in the stem cell-containing limbal region. These results suggest that morphologic transformation of the conjunctival epithelium represents extrinsic environment modulation, and that differential expression of K12 but not K3 keratin signifies corneal epithelial differentiation.
Current Eye Research 11/1994; 13(10):765-78. DOI:10.3109/02713689409047012 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The full-length cDNA of mouse K12 keratin was characterized by sequencing overlapping cDNA clones isolated from a mouse cornea cDNA library. Using Northern blot hybridization, the radio-labeled cDNA hybridized to a 1.9 kb mRNA from adult cornea, but not from other mouse tissues including snout, esophagus, tongue, and skin. During mouse development, corneas do not express K12 mRNA until 4 days postnatal when the epithelium begins to stratify as judged by Northern blot and in situ hybridization. In situ hybridization with 3H-labeled cDNA probe and immunohistochemical studies with antibodies against a synthetic oligo-peptide deduced from rabbit K12 cDNA demonstrate that this mouse K12 keratin is expressed in all cell layers of adult corneal epithelium, and the suprabasal layers, but not the basal layer of the limbal epithelium. Epidermal growth factor (EGF) has been shown to promote epithelium stratification of cultured chicken and human corneas in vitro. To examine whether EGF can promote K12 expression, EGF was administered to neonatal mice. The results indicate that EGF retards K12 expression by corneal epithelial cells, even though it promotes corneal epithelial stratification during mouse development. Taken together, our results demonstrate that the expression of K12 keratin is cornea-specific, differentiation-dependent, and developmentally regulated.
Current Eye Research 12/1993; 12(11):963-74. DOI:10.3109/02713689309029222 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Northern blot hybridization, reverse-transcription polymerase chain reaction (RT-PCR), and RNase protection assays were used to examine the expression of two alpha 1(IX) collagen mRNA species (long and short form) in developing mouse tissues. Furthermore, in situ hybridization was used to identify cells expressing the Col9a1 gene during eye development. The results indicate that during embryonic development eye and heart preferentially express the short form; lung and cartilage express the long form; whereas liver expresses a very low level of long form alpha 1(IX) mRNA which can only be detected by RT-PCR. In situ hybridization demonstrated that at 10.5 day postcoitum (d.p.c.), the alpha 1(IX) collagen mRNAs were first expressed in optic cup (neural ectoderm) but not in lens vesicle (surface ectoderm). By 13.5 d.p.c., the cells that express the alpha 1(IX) mRNA progressively were concentrated toward the anterior part of the neural retina. By 16.5-18.5 d.p.c., the hybridization signals were found exclusively in the inner non-pigmented layer of the presumptive ciliary epithelium. As ciliary epithelial cells become well differentiated 3 weeks after birth, cells expressing the Col9a1 gene were limited to the junction between mature ciliary folds and the neural retina. No hybridization signal could be detected in ocular tissues of mouse older than 6 weeks. It is of interest to note that a hybridization signal was not detected in cornea at the various developmental stages examined, suggesting that mouse cornea does not significantly express alpha 1(IX) mRNA during embryonic development. This differs from that of chick cornea development. In summary, the expression of the Col9a1 gene shows a temporospatial pattern throughout mouse eye development.(ABSTRACT TRUNCATED AT 250 WORDS)