Plasma membrane-cytoskeleton damage in eye lenses of transgenic mice expressing desmin

Immunocytochemistry of eye lens cells from transgenic mice coexpressing desmin and vimentin reveals that the transgenic desmin expression is not uniform. In the same lens, some epithelial and fiber cells overexpress desmin, while in others the desmin gene seems to be silent. Conversely, the endogenous vimentin is always expressed. The concomitant expression of vimentin and desmin results in the assembly of hybrid intermediate filaments (IFs). Moreover, the overexpression of the transgene generates pleomorphic IF assembly and leads to intermingled filamentous whorls and to accumulation of amorphous desmin. The abnormalities of IF assembly induced by the genetic manipulation are correlated with perturbation of the enucleation process in the lens fibers, changes in cell shape, fiber fusion and extensive internalization of the general plasma membrane and junctional domains. The alterations of lens cells described in this study were observed in all transgenic mice examined. The level of expression of the transgene was paralleled by the degree of damage. Our results indicate that proper expression, assembly and membrane interaction of IFs play an important role in the terminal differentiation of the lenticular epithelium into fiber cells. We anticipate that alterations during these processes may initiate cataract formation.     

A role for lengsin, a recruited enzyme, in terminal differentiation in the vertebrate lens

Lengsin is an eye lens-specific member of the glutamine synthetase (GS) superfamily. Lengsin has no GS activity, suggesting that it has a structural rather than catalytic role in lens. In situ hybridization and immunofluorescence showed that lengsin is expressed in terminally differentiating secondary lens fiber cells. Yeast two-hybrid (Y2H) and recombinant protein experiments showed that full-length lengsin can bind the 2B filament region of vimentin. In affinity chromatography, lengsin also bound the equivalent region of CP49 (BFSP2; phakinin), a related intermediate filament protein specific to the lens. Both the vimentin and CP49 2B fragments bound lengsin in surface plasmon resonance spectroscopy with fast association and slow dissociation kinetics. Lengsin expression correlates with a transition zone in maturing lens fiber cells in which cytoskeleton is reorganized. Lengsin and lens intermediate filament proteins co-localize at the plasma membrane in maturing fiber cells. This suggests that lengsin may act as a component of the cytoskeleton itself or as a chaperone for the reorganization of intermediate filament proteins during terminal differentiation in the lens.     

Wnt signaling is required for organization of the lens fiber cell cytoskeleton and development of lens three-dimensional architecture

How an organ develops its characteristic shape is a major issue. This is particularly critical for the eye lens as its function depends on having appropriately ordered three-dimensional cellular architecture. Recent in vitro studies indicate that Wnt signaling plays key roles in regulating morphological events in FGF-induced fiber cell differentiation in the mammalian lens. To further investigate this the Wnt signaling antagonist, secreted frizzled-related protein 2 (Sfrp2), was overexpressed in lens fiber cells of transgenic mice. In these mice fiber cell elongation was attenuated and individual fibers exhibited irregular shapes and consequently did not align or pack regularly; microtubules, microfilaments and intermediate filaments were clearly disordered in these fibers. Furthermore, a striking feature of transgenic lenses was that fibers did not develop the convex curvature typically seen in normal lenses. This appears to be related to a lack of protrusive processes that are required for directed migratory activity at their apical and basal tips as well as for the formation of interlocking processes along their lateral margins. Components of the Wnt/Planar Cell Polarity (PCP) pathway were downregulated or inhibited. Taken together this supports a role for Wnt/PCP signaling in orchestrating the complex organization and dynamics of the fiber cell cytoskeleton.     

Ras signaling is essential for lens cell proliferation and lens growth during development

The vertebrate ocular lens is a simple and continuously growing tissue. Growth factor-mediated receptor tyrosine kinases (RTKs) are believed to be required for lens cell proliferation, differentiation and survival. The signaling pathways downstream of the RTKs remain to be elucidated. Here, we demonstrate the important role of Ras in lens development by expressing a dominant-negative form of Ras (dn-Ras) in the lens of transgenic mice. We show that lens in the transgenic mice was smaller and lens growth was severely inhibited as compared to the wild-type lens. However, the lens shape, polarity and transparency appeared normal in the transgenic mice. Further analysis showed that cell proliferation is inhibited in the dn-Ras lens. For example, the percentage of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in epithelial layer was about 2- to 3-fold lower in the transgenic lens than in the wild-type lens, implying that Ras activity is required for normal cell proliferation during lens development. We also found a small number of apoptotic cells in both epithelial and fiber compartment of the transgenic lens, suggesting that Ras also plays a role in cell survival. Interestingly, although there was a delay in primary fiber cell differentiation, secondary fiber cell differentiation was not significantly affected in the transgenic mice. For example, the expression of beta- and gamma-crystallins, the marker proteins for fiber differentiation, was not changed in the transgenic mice. Biochemical analysis indicated that ERK activity, but not Akt activity, was significantly reduced in the dn-Ras transgenic lenses. Overall, our data imply that the RTK-Ras-ERK signaling pathway is essential for cell proliferation and, to a lesser extent, for cell survival, but not for crystallin gene expression during fiber differentiation. Thus, some of the fiber differentiation processes are likely mediated by RTK-dependent but Ras-independent pathways.     

Transgenic expression of the muscle-specific intermediate filament protein desmin in nonmuscle cells

The coding region of the hamster desmin gene was fused to the 5' flanking sequences of the hamster vimentin gene and introduced into the germ line of mice. The expression of this intermediate filament gene construct (pVDes) was analyzed at the RNA and protein level in transgenic mice as well as in fibroblast cell lines and primary hepatocyte cultures derived from these mice. In all transgenic mice, the pVDes-encoded protein was coexpressed with mouse vimentin in a tissue-specific fashion and was indistinguishable from normal hamster desmin. Culturing of transgenic hepatocytes induced desmin expression indicating that 3.2 kbp of the vimentin gene 5' region regulates both tissue-specific and tissue culture-induced intermediate filament protein expression. Immunohistochemical staining and double-label immunoelectron microscopy of cultured transgenic fibroblasts showed that the pVDes protein assembled into intermediate filaments which colocalized with the mouse vimentin filaments. Endogenous vimentin RNA levels were not influenced by high-level pVDes expression. The coexpression of desmin and vimentin in nonmuscle cells did not result in detectable developmental, morphological, or physiological abnormalities.     

Filensin: a new vimentin-binding, polymerization-competent, and membrane-associated protein of the lens fiber cell

We have studied the molecular properties of a 100-kD protein, termed filensin, which we have isolated from porcine lens membranes. Filensin represents a membrane-associated element, resistant to salt and nonionic detergent treatment, and extractable only by alkali or high concentrations of urea. By indirect immunofluorescence and immunoelectron microscopy, this protein can be localized at the periphery of the lens fiber cells. Immunochemical analysis suggests that filensin originates from a larger 110-kD component which is abundantly expressed in lens but not in other tissues. Purified filensin polymerizes in a salt-dependent fashion and forms irregular fibrils (integral of 10 nm in diameter) when reconstituted into buffers of physiological ionic strength and neutral pH. Radiolabeled filensin binds specifically to lens vimentin under isotonic conditions, as demonstrated by affinity chromatography and ligand-blotting assays. By the latter approach, filensin also reacts with a 47-kD peripheral membrane protein of the lens cells. Purified filensin binds to PI, a synthetic peptide modelled after a segment of the COOH-terminal domain of peripherin (a type III intermediate filament protein highly homologous to vimentin), but not to various other peptides including the NH2-terminal headpiece of vimentin and derivatives of its middle (rod) domain. The filensin-PI binding is inhibited by purified lamin B, which is known to interact in vitro with PI (Djabali, K., M.-M. Portier, F. Gros, G. Blobel, and S. D. Georgatos. 1991. Cell. 64:109-121). Finally, limited proteolysis indicates that the filensin-vimentin interaction involves a 30-kD segment of the filensin molecule. Based on these observations, we postulate that the lens fiber cells express a polymerization-competent protein which is tightly associated with the plasma membrane and has the potential to serve as an anchorage site for vimentin intermediate filaments.     

Lens Fiber Cell Differentiation and Denucleation Are Disrupted through Expression of the N-Terminal Nuclear Receptor Box of Ncoa6 and Result in p53-dependent and p53-independent Apoptosis

Nuclear receptor coactivator 6 (NCOA6) is a multifunctional protein implicated in embryonic development, cell survival, and homeostasis. An 81-amino acid fragment, dnNCOA6, containing the N-terminal nuclear receptor box (LXXLL motif) of NCOA6, acts as a dominant-negative (dn) inhibitor of NCOA6. Here, we expressed dnNCOA6 in postmitotic transgenic mouse lens fiber cells. The transgenic lenses showed reduced growth; a wide spectrum of lens fiber cell differentiation defects, including reduced expression of γ-crystallins; and cataract formation. Those lens fiber cells entered an alternate proapoptotic pathway, and the denucleation (karyolysis) process was stalled. Activation of caspase-3 at embryonic day (E)13.5 was followed by double-strand breaks (DSBs) formation monitored via a biomarker, γ-H2AX. Intense terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) signals were found at E16.5. Thus, a window of ∼72 h between these events suggested prolonged though incomplete apoptosis in the lens fiber cell compartment that preserved nuclei in its cells. Genetic experiments showed that the apoptotic-like processes in the transgenic lens were both p53-dependent and p53-independent. Lens-specific deletion of Ncoa6 also resulted in disrupted lens fiber cell differentiation. Our data demonstrate a cell-autonomous role of Ncoa6 in lens fiber cell differentiation and suggest novel insights into the process of lens fiber cell denucleation and apoptosis.     

Characterization of lens fiber cell triton insoluble fraction reveals ERM (ezrin, radixin, moesin) proteins as major cytoskeletal-associated proteins

To understand lens fiber cell elongation- and differentiation-associated cytoskeletal remodeling, here we identified and characterized the major protein components of lens fiber cell Triton X-100 insoluble fraction by mass spectrometry and immunoblot analysis. This analysis identified spectrin, filensin, vimentin, tubulin, phakinin, and β-actin as major cytoskeletal proteins in the lens fibers. Importantly, ezrin, radixin, and moesin (ERM), heat-shock cognate protein 70, and β/γ-crystallins were identified as major cytoskeletal-associated proteins. ERM proteins were confirmed to exist as active phosphorylated forms that exhibited intense distribution in the organelle free-zone fibers. Furthermore, ERM protein phosphorylation was found to be dramatically reduced in Rho GTPase-targeted transgenic mouse lenses. These data identify the ERM proteins, which cross-link the plasma membrane and actin, as major and stable cytoskeletal-associated proteins in lens fibers, and indicate a potential role(s) for the ERMs in fiber cell actin cytoskeletal and membrane organization.     

Activated Ras induces lens epithelial cell hyperplasia but not premature differentiation

Growth factor signaling is implicated in the regulation of lens cell proliferation and differentiation during development. Activation of growth factor receptor tyrosine kinases is known to activate Ras proteins, small GTP-binding proteins that function as part of the signal transduction machinery. In the present study, we examined which classical Ras genes are expressed in lens cells during normal development and whether expression of an activated version of Ras is sufficient to induce either lens cell proliferation or fiber cell differentiation in transgenic mice. In situ hybridization showed H-Ras, K-Ras and N-Ras are ubiquitously expressed in all cells of the embryonic (E13.5) eye, with N-Ras showing the highest level of expression. The expression level of N-Ras decreases during later stages of embryonic development, and is nearly undetected in postnatal day 21 lenses. To generate transgenic mice, a constitutively active H-Ras mutant was linked to a chimeric regulatory element containing the mouse alphaA-crystallin promoter fused to the chick delta1-crystallin lens enhancer element. In the lenses of the transgenic mice, the transgene was expressed in both lens epithelial and fiber cells. Expression of activated Ras was sufficient to stimulate lens cell proliferation but not differentiation, implying that alternative or additional signal transduction pathways are required to induce fiber cell differentiation.     

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.     

The retinoblastoma protein-binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice.

Regulation of the cell cycle is a critical aspect of cellular proliferation, differentiation, and transformation. In many cell types, the differentiation process is accompanied by a loss of proliferative capability, so that terminally differentiated cells become postmitotic and no longer progress through the cell cycle. In the experiments described here, the ocular lens has been used as a system to examine the role of the retinoblastoma protein (pRb) family in regulation of the cell cycle during differentiation. The ocular lens is an ideal system for such studies, since it is composed of just two cell types: epithelial cells, which are capable of proliferation, and fiber cells, which are postmitotic. In order to inactivate pRb in viable mice, genes encoding either a truncated version of simian virus 40 large T antigen or the E7 protein of human papillomavirus were expressed in a lens-specific fashion in transgenic mice. Lens fiber cells in the transgenic mice were found to incorporate bromodeoxyuridine, implying inappropriate entry into the cell cycle. Surprisingly, the lens fiber cells did not proliferate as tumor cells but instead underwent programmed cell death, resulting in lens ablation and microphthalmia. Analogous lens alterations did not occur in mice expressing a modified version of the truncated T antigen that was mutated in the binding domain for the pRb family. These experimental results indicate that the retinoblastoma protein family plays a crucial role in blocking cell cycle progression and maintaining terminal differentiation in lens fiber cells. Apoptotic cell death ensues when fiber cells are induced to remain in or reenter the cell cycle.     

Bmp signaling is required for development of primary lens fiber cells

We have investigated the role of Bmp signaling in development of the mouse lens using three experimental strategies. First, we have shown that the Bmp ligand inhibitor noggin can suppress the differentiation of primary lens fiber cells in explant culture. Second, we have expressed a dominant-negative form of the type 1 Bmp family receptor Alk6 (Bmpr1b -- Mouse Genome Informatics) in the lens in transgenic mice and shown that an inhibition of primary fiber cell differentiation can be detected at E13.5. Interestingly, the observed inhibition of primary fiber cell development was asymmetrical and appeared only on the nasal side of the lens in the ventral half. Expression of the inhibitory form of Alk6 was driven either by the alpha A-cystallin promoter or the ectoderm enhancer from the Pax6 gene in two different transgenes. These expression units drive transgene expression in distinct patterns that overlap in the equatorial cells of the lens vesicle at E12.5. Despite the distinctions between the transgenes, they caused primary fiber cell differentiation defects that were essentially identical, which implied that the equatorial lens vesicle cells were responding to Bmp signals in permitting primary fiber cells to develop. Importantly, E12.5 equatorial lens vesicle cells showed cell-surface immunoreactivity for bone-morphogenetic protein receptor type 2 and nuclear immunoreactivity for the active, phosphorylated form of the Bmp responsive Smads. This indicated that these cells had the machinery for Bmp signaling and were responding to Bmp signals. We conclude that Bmp signaling is required for primary lens fiber cell differentiation and, given the asymmetry of the differentiation inhibition, that distinct differentiation stimuli may be active in different quadrants of the eye.     

Cytoskeletal and contractile structures in bovine lens cell differentiation

Bovine lenses from animals of different ages were separated into two epithelial sections, a cortical region and the lens nucleus. Both the 10000 g supernatant fraction and pellet of these sections were analysed by electrophoresis in SDS-containing polyacrylamide gels. When comparing total protein patterns of the cytoskeletal preparations from the different parts of lenses of different ages a decrease in the amount of vimentin, the protein subunit of lens intermediate-sized filaments (IF), was observed upon lens cell differentiation and aging. Amounts of monomeric (G) and filamentous (F) actin in the different stages of lens cell differentiation were quantitated using the DNase I inhibition technique. A significant increase in the relative amount of F-actin was observed upon fibre cell formation. A slight, but significant increase in the total amount of actin relative to the total amount of cellular protein was observed when passing from the central part of the lens epithelium to the epithelial cells in the elongation zone. In the fibre cells the amount of total actin decreased from cortex to nucleus. A possible function of microfilament-assembly in the process of lens cell differentiation is suggested.     

Temporal regulation of VEID-7-amino-4-trifluoromethylcoumarin cleavage activity and caspase-6 correlates with organelle loss during lens development

Lens fiber cell differentiation involves extensive reconstruction of the cell's architecture, including the degradation and elimination of all membrane-bound organelles via a process that has been likened to apoptosis. Using caspase reporter assays under conditions in which nonspecific cleavage of the reporter peptides by the proteasome has been inhibited, we investigated whether any specific caspase activities are temporally correlated with this process of organelle loss. Extracts from neonatal mouse lenses contained strong VEID-7-amino-4-trifluoromethylcoumarin (AFC) and minor IETD-AFC and LEVD-AFC cleavage activities, but no DEVD-AFC cleavage activity. Further testing suggested that the VEID-AFC and IETD-AFC cleavage activities were likely due to the same enzyme. In lens extracts from rat embryos, VEID-AFC cleavage activity increased during the period when organelles are eliminated, between embryonic days 15.5 and 18.5, whereas procaspase-6 protein levels decreased, suggesting that this enzyme is responsible for VEID-AFC cleavage. By contrast, in extracts from alpha AE7 transgenic mouse lenses in which apoptosis was induced, strong DEVD-AFC cleavage activity and activated caspase-3 protein were detected. Thus, within the same tissue, different caspase activities can predominate depending on the context, normal differentiation versus apoptosis. These results highlight the difference between normal fiber cell differentiation and apoptosis and the capacity of the lens to differentially regulate these two processes.     

Lens calcium activated proteinase: Degradation of vimentin

The lens has been shown to contain a Ca⁺² activated proteinase specific for vimentin. The proteinase is present in the soluble fraction of the cortex but not in the epithelium. It is suggested that this proteinase is expressed during terminal differentiation of the epithelial cells and may be responsible for degradation of the intermediate filaments in the fiber cells. The proteinase is inhibited by EGTA but not by several proteinase inhibitors.     

Differentiation and oncogenesis: phenotypically distinct lens tumors in transgenic mice

We report on two lines of transgenic mice that express a murine alpha A-crystallin/SV40 tumor antigen fusion gene in the eye lens. The alpha T1 line develops fast growing, poorly differentiated lens tumors, whereas the alpha T2 line produces lens tumors that are slow growing and well differentiated. There is a striking difference between these two lines in the temporal and spatial patterns of tumor antigen expression during initial lens development. In the alpha T1 line, the transgene is expressed very early in development in most lens cells, and no primary fiber differentiation takes place. In the alpha T2 line, transgene expression occurs after primary fiber formation has been initiated, and is restricted to differentiating fiber cells. The anterior epithelium from both alpha T lines undergoes normal development and remains morphologically normal until after birth, although in alpha T1 mice, these anterior cells produce considerable amounts of SV40 tumor antigens. This suggests that the state of differentiation of the lens cell plays an important role in its response to oncogene products.     

Increased sensitivity of various genes to endogenous DNase activity in terminal differentiating chick lens fibers

In the lens, epithelial cells from the equatorial zone differentiate into postmitotic elongated fibers. One aspect of this differentiation is nuclear shape transformation and DNA degradation. This process is controlled by DNase activity which in fiber nuclei increases with development. DNase activity is also present in the epithelial cell nuclei which appears to be non-functional but could be activated in vitro by exogenous addition of Ca2+. We have analyzed the possible selective action of endogenous DNase on 3 genes involved in lens terminal differentiation, namely delta-crystallin, beta-tubulin and vimentin, and on 1 gene not thought to participate in this process, ovalbumin. We have compared restriction DNA patterns of these genes in nuclei isolated from 11-day-old chick embryos and incubated in Ca2+-free medium or in fresh epithelial and fiber lens tissue at 11 and 18 days of development. During incubation in vitro of 11-day fiber nuclei, there is a net increase in the sensitivity of the delta-crystallin, beta-tubulin, ovalbumin and vimentin chromatin to the endogenous DNase. The vimentin gene appears to be more stable than the beta-tubulin and delta-crystallin genes indicating a degree of specificity of the endogenous DNase activity. In the epithelial nuclei, the lens-specific genes appear to be more stable but paradoxically there is a net degradation of the ovalbumin gene. In freshly isolated tissues the 4 genes were detected in epithelial and fiber cells at 11 and 18 days. Furthermore, in the mature fibers in which the nuclei were degenerating, the latter genes were still not completely digested.     

Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency.     

Kynurenine inhibits fibroblast growth factor 2-mediated expression of crystallins and MIP26 in lens epithelial cells

Fibroblast growth factor-2 (FGF2)-mediated signaling plays an important role in fiber cell differentiation in eye lens. We had previously shown that kynurenine (KYN) produced from the overexpression of indoleamine 2,3-dioxygenase (IDO) causes defects in the differentiation of fiber cells, induces fiber cell apoptosis and cataract formation in the mouse lens, and leads to cell cycle arrest in cultured mouse lens epithelial cells (mLEC). In this study, we demonstrate that exogenous KYN reduces FGF2-mediated expression of α-, β-, and γ-crystallin and MIP26 in mLEC. We show that endogenously produced KYN in mLEC of IDO transgenic animals causes similar defects in FGF2-induced protein expression and that a competitive inhibitor of IDO prevents such defects. Our data also show that KYN inhibits FGF2-induced Akt and ERK1/2 phosphorylation in mLEC, which are required for crystallin and MIP26 expression in the lens. KYN does not inhibit FGF2 binding to cells but inhibit phosphorylation of FGFR1in mLEC. Together our data suggest that KYN might inhibit FGF2-mediated fiber cell differentiation by preventing expression of crystallins and MIP26. Our studies provide a novel mechanism by which KYN can exert deleterious effects in cells.