Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

MEKK1-deficient mice show an eye open at birth phenotype caused by impairment in embryonic eyelid closure. MEK kinase 1 (MEKK1) is highly expressed in the growing tip of the eyelid epithelium, which displays loose cell-cell contacts and prominent F-actin fibers in wild-type mice, but compact cell contacts, lack of polymerized actin and a concomitant impairment in c-Jun N-terminal phosphorylation in MEKK1-deficient mice. In cultured keratinocytes, MEKK1 is essential for JNK activation by TGF-beta and activin, but not by TGF-alpha. MEKK1-driven JNK activation is required for actin stress fiber formation, c-Jun phosphorylation and cell migration. However, MEKK1 ablation does not impair other TGF-beta/activin functions, such as nuclear translocation of Smad4. These results establish a specific role for the MEKK1-JNK cascade in transmission of TGF-beta and activin signals that control epithelial cell movement, providing the mechanistic basis for the regulation of eyelid closure by MEKK1. This study also suggests that the signaling mechanisms that control eyelid closure in mammals and dorsal closure in Drosophila are evolutionarily conserved.

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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.Rapid re-epithelialization is essential for restoration of homeostasis in injured tissues; impaired healing of injured epithelium increases the risks of infection and further damage underlying tissues (1, 2). The cornea provides an ideal model to evaluate interactions of migrating epithelial cells and the extracellular matrix of the underlying basement membrane during wound healing because epithelial injuries of the avascular corneal tissue heal in a bloodless wound field. Various specific proteins such as vinculin (3), keratins (4), CD44 hyaluronan receptors (5), and gelatinases and metalloproteinase inhibitors (6, 7) are up-regulated during corneal epithelial wound healing. These proteins are believed to modulate cell adhesion or migration.Lumican belongs to the family of small leucine-rich proteoglycans (SLRPs) 1 that includes keratocan, mimecan, decorin, biglycan, fibromodulin, epiphycan, and osteoadherin. In the cornea, lumican, keratocan, and mimecan are modified with keratan sulfate glycosaminoglycan chains comprising the keratan sulfate proteoglycans (KSPG) of the stromal extracellular matrix (8 -13). In normal unwounded mouse cornea, lumican mRNA is expressed in stromal keratocytes (14). Lumican KSPG is a key regulator of collagen fibrillogenesis, a process critical to corneal transparency. Mice lacking lumican show an age-dependent corneal opacity and a high proportion of abnormally thick collagen fibers in the corneal stroma (15).Lumican is also widely present as a non-or low-sulfated glycoprotein in connective tissues of many other organ systems, e.g. skeleton, heart, kidney, and lung (14, 16 -18). During mouse embryonic ocular development, lumican is synthesized by keratocytes; detected as a glycoprotein, not as a KSPG (19); and also transiently expressed by the corneal epithelium, neural retina, and epidermis (14). These observations suggest that epithelial tissues possess the capacity to express lumican under certain conditions. Several studies have demonstrated that SLRP proteins can modulate cellular behaviors, i.e. cell migration and prolifera...

BRCAl is proposed to be a tumor suppressor gene. To explore the biological function of BRCAl, a partial deletion (amino acids 300-361) of mouse Brcal exon 11 was introduced into the genome of embryonic stem (ES) cells by homologous recombination. Mice carrying one mutated allele of Brcal appear normal and are fertile up to 10 months of age without any sign of illness. However, no viable progeny homozygous for the Brcal mutant allele were obtained. Detailed analysis of large numbers of embryos at different stages of development indicated that the homozygous mutant concepti are severely retarded in growth as early as embryonic day 4.5 (E4.5) and are resorbed completely by E8.5. Although the homozygotes at E5.5-E6.5 are able to synthesize DNA and display distinguishable embryonic and extraembryonic structures, they fail to differentiate and form egg cylinders. Consequently, they were unable to form primitive streaks and undergo gastrulation. Consistent with these in vivo results, blastocysts homozygous for mutated Brcal alleles are at a considerable disadvantage when grown in vitro. These observations suggest that Brcal has an important role in the early development of mouse embryos.

A gene assigned to human chromosome 1q32-41 encodes a novel protein of 3,113 amino acids containing an internal tandem repeat of 177 amino acids. The protein, which we have named ''mitosin,'' was identified by direct binding to purified retinoblastoma protein in vitro with a region distantly related to the retinoblastoma protein-binding site of E2F-1. Mitosin is expressed throughout S, G 2 , and M phases of the cell cycle but is absent in G 1 . Its localization is dramatically reorganized from a rather homogeneous nuclear distribution in S phase to paired dots at the kinetochore/centromere region, to the spindle apparatus, and then to the midbody during M-phase progression. This spatial reorganization coincides closely with the temporal phosphorylation patterns of mitosin. Overexpression of N-terminally truncated mutants blocks cell cycle progression mainly at G 2 /M. These results suggest that mitosin may play an important role in mitotic-phase progression.

Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera ؊/؊ cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera ؊/؊ cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera ؊/؊ mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision.The vertebrate cornea is a tough and transparent tissue that provides greater than 60% of refractive power to the incoming light to cast a focused image on retina. To exact its proper functions, the cornea has to maintain transparent, appropriate curvature for refraction and toughness for protection. Genetic and epigenetic factors that cause the change of corneal curvature, reduction of transparency, and weakness in structure can lead to the impairment of visual acuity.The corneal stroma consists of uniformly small collagen fibrils with an average diameter of 25 nm that are arranged in orthogonal lamellae. Stromal keratocytes are responsible for the formation and maintenance of a unique collagenous matrix that is essential for proper corneal curvature and transparency (1, 2). The mechanisms that govern the assembly of different levels of stromal architecture are not well understood; however, proteoglycan-collagen and collagen-collagen interactions have been implicated. It has been suggested that the stoichiometry and interaction of different collagen types play an important role in modulating collagen fibril diameter (1, 3-5). The proteoglycans in the stroma are members of the small leucine-rich proteoglycan (SLR...

To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.