Lens Cortical Fiber Cells Research Articles

Bone morphogenetic protein-4 and bone morphogenetic protein receptors expressions in the adult rat eye.

We investigated the expressions of bone morphogenetic protein-4 (BMP4) and its receptors, bone morphogenetic protein receptor IA (BMPRIA), bone morphogenetic protein receptor IB (BMPRIB) and bone morphogenetic protein receptor II (BMPRII) in the adult rat eye. Interesting differences in expression profile were observed between BMPRIA and BMPRIB in the retina. BMPRIA-like immunoreactivity (IR) was very intensely seen in the photoreceptor layer, while BMPRIB-IR was mainly observed in the other layers. In the cornea, BMP4, BMPRIA, BMPRIB and BMPRII-IRs were abundantly seen in the cell body of basal cells in the corneal epithelium, and endothelium. In the lens, BMP4, BMPRIA, BMPRIB and BMPRIIIRs were observed in epithelial cells, lens cortical fiber cells, however they were not seen in the capsule and the central region of the lens. In the iris and ciliary body, strong BMP4 and BMPRIB-IRs were observed in nonpigmented epithelium. These results suggest that different kinds of BMP signaling should be needed in different areas in the adult eye to keep the shapes, differentiation levels, and functions of various cells.

Age-related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes.

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old-age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C-terminus with significantly higher frequency compared to other residues, suggesting that trypsin-like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA-crystallin peptide (αA(57-65)) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA(57-65) formation escalated significantly. Using 2D gel electrophoresis/nanoLC-ESI-MS/MS, 12 protein complexes of 40-150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross-linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes.

Truncation, cross-linking and interaction of crystallins and intermediate filament proteins in the aging human lens

The optical properties of the lens are dependent upon the integrity of proteins within the fiber cells. During aging, crystallins, the major intra-cellular structural proteins of the lens, aggregate and become water-insoluble. Modifications to crystallins and the lens intermediate filaments have been implicated in this phenomenon. In this study, we examined changes to, and interactions between, human lens crystallins and intermediate filament proteins in lenses from a variety of age groups (0–86years). Among the lens-specific intermediate filament proteins, filensin was extensively cleaved in all postnatal lenses, with truncated products of various sizes being found in both the lens cortical and nuclear extracts. Phakinin was also truncated and was not detected in the lens nucleus. The third major intermediate filament protein, vimentin, remained intact in lens cortical fiber cells across the age range except for an 86year lens, where a single ~49kDa breakdown product was observed. An αB-crystallin fusion protein (maltose-binding protein-αB-crystallin) was found to readily exchange subunits with endogenous α-crystallin, and following mild heat stress, to bind to filensin, phakinin and vimentin and to several of their truncated products. Tryptic digestion of a truncated form of filensin suggested that the binding site for α-crystallin may be in the N-terminal region. The presence of significant amounts of small peptides derived from γS- and βB1-crystallins in the water-insoluble fraction of the lens indicates that these interact tightly with cytoskeletal or membrane components. Interestingly, water-soluble complexes (~40kDa) contained predominantly γS- and βB1-crystallins, suggesting that cross-linking is an alternative pathway for modified β- and γ-crystallins in the lens.

αA-Crystallin associates with α6 integrin receptor complexes and regulates cellular signaling

α-Crystallins are small heat-shock proteins important to lens transparency that provide the lens with its refractive properties. In their role as molecular chaperones, these crystallins also prevent protein aggregation, affect cytoskeletal remodeling, enhance resistance to cell stress, and provide lens cells with protection against apoptosis. While many of the functions assigned to αA-crystallin are attributable to its presence in the cytoplasm of lens cells, αA-crystallin also has been detected at the lens plasma membrane. However, how αA-crystallin becomes linked to the plasma membrane or what its functions are at this site has remained unknown. In this study, we examined the mechanisms by which αA-crystallin becomes associated with the lens membrane, focusing specifically on its interaction with membrane receptors, and the differentiation-specificity of these interactions. We also determined how the long-term absence of αA-crystallin alters receptor-linked signaling pathways. αA-crystallin association with membrane receptors was determined by co-immunoprecipitation analysis; its membrane localization was examined by confocal imaging; and the effect of αA-crystallin loss-of-function on the activation state of signaling molecules in pathways linked to membrane receptors was determined by immunoblot analysis. The results show that, in lens epithelial cells, plasma membrane αA-crystallin was primarily localized to apicolateral borders, reflecting the association of αA-crystallin with E-cadherin complexes. These studies also provide the first evidence that αA-crystallin maintained its association with the plasma membrane in lens cortical fiber cells, where it was localized to lateral interfaces, and further show that this association was mediated, in part, by αA-crystallin interaction with α6 integrin receptor complexes. We report that the absence of αA-crystallin led to constitutive activation of the stress kinases p38 and JNK, classical inducers of apoptotic cell death, and the loss of the phospho-Bad pro-survival signal, effects that were greatest in differentiating lens fiber cells. Concurrent with this, activation of FAK and ERK kinases was increased, demonstrating that these receptor-linked pathways also were dysregulated in the absence of αA-crystallin. These data link αA-crystallin plasma membrane association to its differentiation-state-specific interaction with E-cadherin and α6 integrin receptor complexes. The changes in cell signaling in αA-crystallin-null lenses suggest that dysregulation of receptor-linked cell-signaling pathways that accompany the failure of αA-crystallin to associate with membrane receptors may be responsible for the induction of apoptosis. The observed changes in lens cell signaling likely reflect long-term functional adaptations to the absence of the αA-crystallin chaperone/small heat-shock protein.

Expression and cellular localization of melatonin‐synthesizing enzymes in the rat lens

Melatonin (N-acetyl-5-methoxytryptamine) prevents oxidative stress-induced cataract development, and previous studies have suggested that the ocular lens synthesizes melatonin. In the present study, we examined whether two key enzymes in melatonin biosynthesis, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), are expressed in the lens of adult male rats. Reverse transcriptase-polymerase chain reaction analyses demonstrated that both AANAT and HIOMT mRNAs are expressed in the lens. Western blotting for AANAT protein showed that the lens, like the pineal gland, contains this enzyme protein with a molecular mass of 24 kDa. Immunohistochemistry revealed that AANAT protein is localized to the lens cortical fiber cells. Serotonin, which is a substrate for AANAT and a melatonin precursor, was also found in this region. These findings demonstrate that the lens expresses AANAT and HIOMT, and suggest that the cortical fiber cells are the main melatonin-synthesizing sites in the lens. Locally synthesized melatonin in the lens cortical fiber cells may protect the lens itself from cataract development.

Distribution of caveolin-1 in bovine lens and redistribution in cultured bovine lens epithelial cells upon confluence

The distribution of caveolin-1 in the lens and lens epithelial cells was determined to assess possible roles in cholesterol trafficking, cell to cell communication and signal transduction. Bovine lenses and cultured bovine lens epithelial cells (BLEC) were divided into subcellular fractions and the distribution of proteins recognized by three different caveolin-1 antibodies determined. The immunolocalization of caveolin-1 in the lens epithelium and in subconfluent and confluent cultured BLEC was probed by fluorescence microscopy and laser scanning confocal microscopy. EGF induced phosphorylation of caveolin-1 was detected by Western blotting with an anti-phosphotyrosine antibody to immunoprecipitated caveolin-1 from BLEC and human cancer cells. Monomeric caveolin-1 of about 26 kDa was detected in the epithelial cell membrane of cultured BLEC and fresh epithelia and in the plasma membrane fraction of lens cortical fiber cells. Caveolin-1 of cultured BLEC redistributed from the cytoplasm to plasma membrane as the cells proceeded from subconfluent to confluent states. The apparent abundance of caveolin-1 in cortical fiber cell plasma membrane is consistent with possible roles in distribution of lens membrane cholesterol and membrane structure. The presence of caveolin-1 in the plasma membrane of epithelial cells at – but not before – confluency is consistent with a role of caveolin-1 in cell to cell communications. EGF stimulated phosphorylation of caveolin-1 in human A431 cells but not lens cells.

Electron microscopy supports a fibrous substructure for lens intermediate filaments.

The substructure of intermediate filaments from bovine lens cortical fiber cells was investigated by electron microscopy. Native filaments and synthetic ones regenerated from the total cytoskeletal extract and from the three purified subunits were examined. The morphologies from these various sources were essentially identical, with the exception that filaments reconstituted from one of the purified polypeptides were much shorter, very contorted and showed strings of aggregated protein. The solid cylindrical, unbranching filaments consisted of a helical arrangement of at least two, 5 nm diameter strands. The evidence indicated that each strand was composed of two, 2 nm diameter protofilaments which were also helically constructed (right-handed) with a periodicity of 11.6 nm. Intermediate filament diameter varied widely (8-14.8 nm, average 11.3 nm) and in a direct, linear manner relative to the apparent progression (helical) angle of the strands across the filaments face. These conclusions were obtained from observations on negatively stained intact filaments and reconstituted 4.4 nm fibrils and on positively stained transverse sections of fixed and embedded filaments.