Immunochemical characterization of a Mr 115 lens fiber cell-specific extrinsic membrane protein

Monoclonal and polyclonal antibodies have been produced against a lens fiber cell extrinsic membrane protein, with a relative molecular weight of approximately 115 kd. Enzyme Linked Immunosorbent Assays (ELISA) of retina, ciliary body-iris, liver, and skeletal muscle, utilizing these antibodies, suggest that the antigen is unique to the lens. Immunocytochemistry indicates that the antigen is present only in the differentiated fiber cell, and is absent from the lens epithelium. Further, immunocytochemical reactivity is predominantly associated with the fiber cell plasma membrane. However, sequential extraction of fiber cell homogenate, followed by quantitative, competitive ELISA analysis, indicates that most of the antigen is recovered in the neutral buffer extract. ELISA analysis using monoclonal antibodies indicates that an analogous antigen is present in human and rabbit lenses. On the basis of these results we characterize this antigen as a conserved extrinsic membrane protein, which is unique to the differentiated lens fiber cell. The relationship of this antigen to a previously described Mr 95 beaded filament-associated protein is discussed.     

Discrimination between the lens fiber cell 115 kd cytoskeletal protein and alpha-actinin.

The lens fiber cell cytoskeleton includes a protein with a relative molecular weight, by SDS PAGE, of 115 kD. This protein has been purported to be, or be related to alpha-actinin, a highly-conserved family of actin-binding cytoskeletal proteins common to many tissues across a wide phylogenetic range. In this report we assess the relationship between the 115 kd lens fiber cell protein and alpha-actinin. Assessment of relative molecular weight, immunologic cross-reactivity, and partial sequence analysis suggest that the 115 kD lens fiber cell cytoskeletal protein and alpha-actinin are either unrelated, or, at best, that the lens protein represents an unusually divergent isoform of the alpha-actin family of proteins. Immunochemical analysis of homogenates of bovine heart and red blood cells indicate that these tissues express a protein which is weakly cross-reactive with the lens 115 kD protein, but that this cross-reactive protein is not alpha-actinin.     

Age-related changes in a fiber cell-specific extrinsic membrane protein

Western blot analysis using a monoclonal antibody raised against a lens fiber cell-specific, extrinsic membrane protein reveals several immunologically related bands in fractions derived from bovine lens. Previous work suggests that the parent molecule is the Mr 115 species, and that lower molecular weight bands represent the products of a progressive, step-wise, post-translational degradation. In this report we compare the extent of proteolytic degradation in extracts prepared from the lens cortex and lens nucleus, using both protease-suppressive and protease-permissive isolation protocols. The results suggest that the observed degradation is a result of in vivo post-translational modification of the Mr 115 antigen, and thus represents physiologic aging of this protein. This analysis also suggests that degradation alters the solubility and/or membrane affinity of this antigen, resulting in a progressive shift to the insoluble phase.     

Research in similarities between EEP34 and lipocortin

EDTA-extractable protein (EEP) is a major extrinsic protein of lens fiber membrane. The EEP of calf lens separated three major bands by SDS-PAGE, and the molecular weights of these major bands were 34 kilodalton (kd.), 33 kd. and 32 kd. Combination of EEP with F-actin and some kinds of phospholipid containing liposomes depends on Ca2+. In two-dimensional gel electrophoresis, some spots of the calf-EEP partially overlapped with spots of lipocortin, which is a substrate for epidermal growth factor receptor/kinase and an inhibitor of phospholipase A2. The 34 kd. protein of the EEP cross-reacted to the antibody prepared against lipocortin of pig's aorta: These results suggest the important function of EEP in lens.     

Immunologic conservation of the fiber cell beaded filament

Lenses were obtained from the eyes of four different classes of Chordates, including Mammalia (rat, mouse, cow, human), Aves (chicken), Amphibia (tiger salamander), and Osteichthyes (steelhead), as well as from one Mollusca (squid). Buffer soluble, urea soluble and urea insoluble fractions were prepared from each, and probed by western blot analysis for the presence of the lens fiber cell 115 and 49 kD beaded filament proteins. Application of both polyclonal and monoclonal antibodies revealed that an immunologic homologue to the bovine fiber cell 115 kD protein is present in all examples of Chordates tested, and that this homologue possessed properties very similar to those of its bovine counterpart. Both monoclonal and polyclonal antibodies revealed an immunologically cross-reactive homologue in squid as well, but suggested that the squid protein had a native molecular weight of closer to 70-80 kD. A monoclonal antibody to the bovine 49 kD beaded filament protein was successful at identifying an immunologic homologue to this protein in mouse, chicken, and tiger salamander. Ultrastructural analysis of rat, human, and fish lenses showed that a beaded filament was present in these lenses, which was indistinguishable from that seen in the bovine lens. In the squid a filamentous, beaded structure was observed, but it differed from that seen in the bovine lens. We conclude from the data presented that the beaded filament, and its constituent proteins, are well-conserved. This data should facilitate the identification of lens cytoskeletal proteins and structure in a wide range of animal models, and establish that probes for these proteins may be of broad applicability.     

Immunocytochemical evidence for an actin-myosin system in lens epithelial cells

Since filamentous actin had been shown earlier to exist in lens epithelial and fiber cells, we inquired whether this could represent a contractile system with myosin and other actin-associated proteins. We resolved this question in freshly removed or organ-cultured rabbit and squirrel lens epithelial whole mounts using immunocytochemical techniques and by immunoblots of extracts separated by electrophoresis. In the former, methods were developed using long fixation times and long incubation in primary antibodies and biotinylated second antibodies visualized by streptavidin immunofluorescence and by diaminobenzidine peroxidase. Myosin was found to be localized along the filamentous rays and at central vertices of polygonal arrays situated at the apices of epithelial cells. It was not clear whether myosin and actin occurred together along the same or adjacent filaments in a bundle. Tubulin and vimentin were found deeper in the cells and were not aligned with actin and myosin filaments. Control lens epithelia treated similarly except for deletion of the primary antibodies showed no staining. As positive controls, pieces of glycerinated sartorius muscle exhibited characteristic cross-banded patterns of actin and myosin when incubated with the same reagents used on the lens epithelium. Denatured extracts of rabbit lens epithelium and of cortical fiber cells separated by electrophoresis and transferred to nitrocellulose paper, stained specifically with the same myosin and tubulin antibodies used in the immunocytochemistry experiments. The molecular weight profile of the myosin polypeptide indicated that lens tissue has myosin II. We conclude that a contractile system exists in lens epithelial and cortical fiber cells, although the function is not understood at this time. We conjecture that the system may act to stabilize lens shape by providing contractile tone.     

Ultrastructural, biochemical, and immunologic evidence of receptor-mediated endocytosis in the crystalline lens

The lens epithelium is essentially the basal layer of the crystalline lens of the eye, an uncommon stratified epithelium. Ions and metabolites present in the aqueous humor gain access to the lens epithelium by diffusion through the lens capsule (the basement membrane of the lens epithelium). Then, it is presumed, the underlying lens fiber cells obtain necessary ions, metabolites, and nutrients through gap junctions conjoining the apical surfaces of the lens epithelial cells from the basal layer with the apical surfaces of elongating fiber cells from upper strata. In this report, correlative morphologic, biochemical, and immunochemical evidence is presented that both lens epithelial and fiber cells use endocytotic and/or transcytotic processes rather than being solely dependent on gap junctions for metabolic cooperation. Freeze-fracture analysis of the apicoapical interface between lens epithelial and elongating fiber cells (epithelial-fiber cell interface [EFI]) revealed protrusions and pits of two distinct sizes (average diameters, 46 and 126 nm). Gap junctions with tight particle packing were only rarely observed at the EFI. Gap junctions with loose particle packing were never observed at the EFI. "Orthogonal arrays" of intramembrane particles (OAPs) were not uncommon at the EFI. Thin-sections taken perpendicular to the EFI confirmed the existence of micropinocytotic and clathrin-coated vesicles in both lens epithelial and elongating fiber cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of separate preparations of lens epithelial and fiber cells, specifically enriched for clathrin-coated vesicles, showed a 180-kD protein. Western blot analysis of this protein revealed selective cross-reactivity with polyclonal anticlathrin antibodies. These results strongly suggest that transcytotic processes provide a primary route for the entry and egress of macromolecules in the lens.     

Disappearance of p27(KIP1) and increase in proliferation of the lens cells after extraction of most of the fiber cells of the lens

PURPOSE: Proliferation of the lens epithelial cells is involved in the fibrotic changes of lens capsules after cataract extraction. However, the mechanisms of the proliferation of the lens epithelial cells are largely unknown. The purpose of this study was to examine the correlation between the expression of p27(KIP1) and cell proliferation in the lens cells after the extraction of lens fiber cells. METHODS: At embryonic days (E) 14 and 18, the C57Bl6 mice were anesthetized and the embryos were surgically removed. The eyes were dissected from these embryos and also from mice 12 weeks after birth. The 12-week-old mice were anesthetized, and then the lens fiber cells were extracted. Normal eyes at E14 and 18 and eyes fixed at 15 min and 48 hr after the extraction of the lens fiber cells were analyzed using immunohistochemistry with anti-p27(KIP1), p57(KIP2), and phosphorylated extracellular signal-regulated kinase (phospho-ERK) 1/2 antibodies, and cells in the S phase of the cell cycle were also examined using anti-bromodeoxyuridine (BrdU) antibody. RESULTS: p27(KIP1) and p57(KIP2)-positive cells were present in the equatorial region of E14 mice. At E18, many lens fiber cells showed nuclear immunoreactivity for p27(KIP1), whereas a small number of cells were positive for p57(KIP2) in the equatorial region. At 12 weeks of age, all nuclei of the lens epithelial cells as well as lens fiber cells showed nuclear immunoreactivity for p27(KIP1). In contrast, p57(KIP2) and phospho-ERK1/2 were not expressed in the lens cells. At 15 min after the extraction of lens fiber cells, phospho-ERK1/2 as well as p27(KIP1) were detected in the lens cells. At 48 hr after the extraction of the lens fiber cells, a few p27(KIP1)-positive nuclei were observed in the equatorial region of the lens capsule. In contrast, many lens cells showed nuclear immunoreactivity for BrdU. CONCLUSIONS: These findings suggest that degradation of p27(KIP1) mediated by phosphorylation of ERK 1/2 is correlated with proliferation of the epithelial cells after the extraction of the lens fiber cells.     

N-cadherin of the human lens

N-cadherin was identified in the human lens by its immunological specificity, and concanavalin-A (Con-A) binding. The 135 kd glycoprotein was partially purified from human lens plasma membranes by Con-A affinity column chromatography. In the newborn lens, N-cadherin is distributed equally in amount between cortical and nuclear membranes. It is markedly decreased in the nuclear membranes of the 2 year-old lens and was no longer detectable in the nucleus of 15 yr-old and older lenses (15 yrs - 86 yrs). Such nuclear loss of N-cadherin is consistent with similar findings in the chicken and bovine lens. At all ages, N-cadherin was readily detected in cortical fiber-cells. When expressed as a ratio to MP26 content, the amount of N-cadherin of the total fiber mass declines at least 4-fold from newborn to 15 years of age, and remains stable thereafter. Homogenization of bovine lenses in the presence of Ca++ resulted in a marked loss of the protein, suggestive of degradation by a calcium-activated protease. The loss of N-cadherin with aging in fiber cells suggests either an alteration in the mode of membrane adhesion of these cells, or a decline in adhesiveness of nuclear as compared to cortical fiber-cells.     

Ferritin H- and L-chains in fiber cell canine and human lenses of different ages

PURPOSE: This study was designed to elucidate potential age-related changes in the concentration, structure, and assembly pattern of ferritin chains in lens fiber cells. METHODS: Canine and human lens fiber cell homogenate proteins were separated by one-dimensional and two-dimensional SDS-PAGE. Ferritin chains were immunodetected and quantitated with ferritin chain-specific antibodies. Total ferritin concentration was measured by ELISA. Binding of iron was determined in vitro with (59)Fe. RESULTS: Ferritin H- and L-chains in canine and human fiber cells of healthy lenses were extensively modified. The H-chain in both species was truncated, and its concentration increased with age. Canine L-chain was approximately 11 kDa larger than standard canine L-chain, whereas human L-chain was of the proper size. Two-dimensional separation revealed age-related polymorphism of human and canine lens fiber cell L-chains and human H-chains. Normal size ferritin chains were not identified in canine fiber cells, but a small amount of fully assembled ferritin was detected, and its concentration decreased with age. CONCLUSIONS: Such significantly altered ferritin chains are not likely to form functional ferritin capable of storing iron. Therefore, lens fiber cells, particularly from older lenses, may have limited ability to protect themselves against iron-catalyzed oxidative damage.     

Immunohistochemical localization of phosphatidylinositol-4,5-bisphosphate in the rat lens

We have attempted to localize immunohistochemically phosphatidylinositol-4,5-bisphosphate (PIP2) in rat lens tissue using affinity purified rabbit anti-PIP2 antibodies. Evidence indicates that PIP2 is localized to the lens epithelial cells but appears to be absent from the lens fiber cells.     

The distribution of the main intrinsic membrane polypeptide in ocular lens

The Main Intrinsic Polypeptide (MIP) of the ocular lens fiber cell plasma membrane was immunocytochemically localized at the ultrastructural level on ultrathin frozen sections of rat lens, and on extracted, gradient-purified bovine lens membranes. The results indicate that both the junctional and non-junctional membrane domains of the cortical lens fiber cell are MIP immunoreactive. Frozen thin section immunocytochemistry of the lens epithelium and hepatocytes, also using anti-MIP antibodies, revealed that these cells, and their intercellular junctions, are not MIP-immunoreactive. From these findings we conclude that 1) MIP, a putative fiber cell junctional protein, is present throughout the plasma membrane of the lens fiber cell, and is not confined to the fiber cell junctional domain, 2) MIP is not a detectable component of the lens epithelial cell membrane, or its intercellular junctions, 3) MIP is not detectable in gap junctions of hepatocytes.     

Phosphatidylinositol 3-kinase in bovine lens and its stimulation by insulin and IGF-1

PURPOSE: To identify and characterize phosphatidylinositol 3-kinase (PI-3K) in the lens and to study its involvement as a signal mediator in lens epithelial cells exposed to insulin and insulin-like growth factor (IGF)-1, which are known to induce lens epithelial cell proliferation and differentiation into fiber cells. METHODS: Concentric fiber cell layers from single bovine lens were prepared by dissolution in buffer. PI-3K activity in capsule-epithelium and fiber cell layers was determined after immunoprecipitation with antibodies against p85, the regulatory subunit of PI-3K. High-performance liquid chromatography on an ion exchange column (Partisil-SAX; Whatman, Maidstone, United Kingdom) was used to identify PI-3K reaction products. Cultured bovine lens epithelial cells were stimulated with insulin or IGF-1, and PI-3K activity was determined after immunoprecipitation with antibody against phosphotyrosine. Association of p85 with other proteins after stimulation was determined in anti-p85 immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis using anti-phosphotyrosine antibody. RESULTS: PI-3K activity was found in both lens epithelial cells and fiber cells. The highest specific activity was found in the capsule-epithelium, but there was considerable activity in other fiber cell layers. Insulin and IGF-1 stimulated the PI-3K activity in epithelial cells in culture by more than 100%, and activation of the enzyme resulted in tyrosine phosphorylation of the p85 subunit. After stimulation, the p85 subunit of PI-3K was associated with 100- and 180-kDa tyrosine phosphorylated proteins. CONCLUSIONS: The activation of PI-3K and its association with specific tyrosine-phosphorylated proteins may be important in insulin and IGF-1 signal transduction in lens epithelial cells. The presence of significant PI-3K activity throughout the lens further suggests that this signal transduction enzyme is sustained in fiber cells.     

Accumulation of thrombospondin-1 in post-operative capsular fibrosis and its down-regulation in lens cells during lens fiber formation

Thrombospondin-1 (TSP-1) is a glycoprotein involved in activation of latent transforming growth factor beta (TGFbeta) expression. We examined changes in its expression pattern during human capsular opacification (PCO) and anterior subcapsular cataractogenesis (ASC), as well as in a healing injured mouse lens. Its expression pattern was also compared in a mouse embryonic lens with that in an adult lens. Based on immunohistochemistry under light microscopy, TSP-1 expression and other matrix components were evident in the anterior epithelium of an uninjured human lens, whereas fiber-differentiating cells in the equator of human lens lack TSP-1 immunoreactivity. In contrast, in post-operative human lens epithelial or fibroblastic cells, there was TSP-1 immunoreactivity, whereas it decreased in fiber-differentiating cells in PCO. Matrix components accumulated on the healing capsule also labeled with anti-TSP-1 antibody like antibodies against collagen I, IV, V and laminin. In uninjured, injured mouse lens epithelial cells and its matrix, there was TSP-1 expression. Embryonic lens cells in the posterior pole, undergoing differentiation to fiber cells, began to express TSP-1 protein at embryonic day (E) 11.5 whereas anterior epithelial cells started to express it at E13.5 in association with marked expression in central fiber cells. At E16.5, TSP-1 was detected in fibers just beneath the anterior epithelium, but the fiber mass showed minimal expression. At E18.5 and post-natally day 1, lens fiber TSP-1 expression was no longer seen. On the other hand, it was evident in both intact human anterior epithelial and dispersed mouse cells. The results indicate that there is TSP-1 expression in uninjured human and mouse lens epithelial cells and their fibrous tissue. In contrast, in post-operative lens cells differentiating to fiber cells, its expression levels decline. Further study is needed to clarify the roles of TSP-1 in modulating lens cell phenotype expression.     

INFLUENCE OF NEURAL RETINA ON LENS FIBRE DIFFERENTIATION IN RATS

Lens epithelial explants grown in retina-conditioned medium (RCM) undergo structural and molecular changes characteristic of fibre differentiation in the intact lens. We suggest that\n vivo neural retina releases a fibre differentiation factor (FDF) that interacts with equatorial lens epithelial cells and stimulates them to undergo fibre cell differentiation. According to this model, interaction with neural retina is essential for normal lens formation in embryos and for normal lens growth throughout life. Preliminary work on purification of the factor indicates that FDF activity is associated with a high molecular weight complex of 500 kd. The active component of this complex appears to be an 80 kd molecule.     

Influence of neural retina on lens fibre differentiation in rats

Lens epithelial explants grown in retina-conditioned medium (RCM) undergo structural and molecular changes characteristic of fibre differentiation in the intact lens. We suggest that in vivo neural retina releases a fibre differentiation factor (FDF) that interacts with equatorial lens epithelial cells and stimulates them to undergo fibre cell differentiation. According to this model, interaction with neural retina is essential for normal lens formation in embryos and for normal lens growth throughout life. Preliminary work on purification of the factor indicates that FDF activity is associated with a high molecular weight complex of 500 kd. The active component of this complex appears to be an 80 kd molecule.     

Expression and activation of the epidermal growth factor receptor in differentiating cells of the developing and post-hatching chicken lens

The epidermal growth factor receptor is hypothesized to play an important role in the post-natal growth and differentiation of the ocular lens. Immunohistochemistry and western blotting were utilized to examine the distribution and activation of the epidermal growth factor receptor in embryonic and post-hatching chicken lenses. Although present at constant levels within epithelial cells throughout embryonic development, the receptor becomes increasingly activated on a highly conserved tyrosine residue necessary for intracellular signal transduction as hatching approaches. After hatching, activated receptors are found in epithelial cells committed to fiber cell formation and in fiber cells undergoing initial stages of terminal differentiation. Activated receptors could not be identified in central epithelial cells or nuclear fiber cells. This pattern persists until at least one year post-hatching. These data indicate that the epidermal growth factor receptor is positioned to influence not only post-natal patterns of lenticular gene expression but also the greatest amount of lens growth and development.     

Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens

PURPOSE: To map changes in the structure and function of fiber cell gap junctions that occur with lens differentiation. METHODS: Equatorial lens sections were fluorescently labeled with antibodies to the gap junction protein connexin (Cx)46, the membrane marker wheat germ agglutinin, and the nuclear stain propidium iodide. Two-photon microscopy and digital image analysis were used to quantify label and cell morphology as a function of radial distance (r/a) across the lens. Loop- and tail-specific Cx46 antibodies were used to identify regions of posttranslational modification. Local fiber cell coupling was imaged in situ using two-photon flash photolysis of caged fluorescein. RESULTS: Antibody labeling showed that the cytoplasmic tail of Cx46 was removed in two zones (r/a approximately 0.9 and r/a approximately 0.7). In addition, with increasing depth, the large radially aligned plaques of peripheral fiber cells became fragmented and dispersed around the cell membrane, and cells became more circular in cross section. Fluorescein transfer between peripheral fiber cells was highly anisotropic and occurred predominantly within a column of fiber cells, resulting in radially directed transport. In regions beyond the zone of nuclear loss, transport was more isotropic and occurred across columns of fiber cells. CONCLUSIONS: The cleavage of Cx46 is associated with a spatial redistribution of gap junction plaques. The distribution of gap junction plaques around the cell membrane can explain the observed directionality of intercellular solute transfer. The findings suggest that the processing and redistribution of gap junction proteins is central to controlling radial and circumferential solute gradients in different regions within the lens.