Postnatal Lens Research Articles

Altered Cell Clusters and Upregulated Aqp1 in Connexin 50 Knockout Lens Epithelium.

To characterize the heterogeneity and cell clusters of postnatal lens epithelial cells (LECs) and to investigate the downstream targets of connexin 50 (Cx50) in the regulation of lens homeostasis and lens growth. To determine differentially expressed genes (DEGs) in the connexin 50 knockout (Cx50KO) lens epithelial cells that shed light on novel mechanism underlying the cataract and small size of the Cx50KO lenses. Single-cell RNA sequencing (scRNA-seq) of lens epithelial cells isolated from one-month-old Cx50KO and wild-type (WT) mice were performed. Differentially expressed genes were identified, and selected DEGs were further studied by quantitative real-time PCR (RT-qPCR) analysis and Western blot analysis. The expression profiles of several thousand genes were identified by scRNA-seq data analysis. In comparison to the WT control, many DEGs were identified in the Cx50KO lens epithelial cells, including growth regulating transcriptional factors and genes encoding water channels. Significantly upregulated aquaporin 1 (Aqp1) gene expression was confirmed by RT-qPCR, and upregulated AQP1 protein expression was confirmed by Western blot analysis and immunostaining both in vivo and in vitro. Lens epithelial cells exhibit an intrinsic heterogeneity of different cell clusters in regulating lens homeostasis and lens growth. Upregulated Aqp1 in Cx50KO lens epithelial cells suggests that both connexin 50 and AQP1 likely play important roles in regulating water homeostasis in lens epithelial cells.

Verification of the gene and protein expression of the aquaglyceroporin AQP3 in the mammalian lens

Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP, AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens. AQP3 was found in all lens regions, with the highest signal of non-glycosylated AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of AQP3. Immunolabelling of lens sections with AQP3 antibodies confirmed that AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.

Requirement for Crk and CrkL during postnatal lens development

The Crk and CrkL adaptor proteins have SH2 and SH3 domains and play essential overlapping, as well as distinct, roles in many biological processes, ranging from cell structure and motility to proliferation. Conditional ablation of both Crk and CrkL in neuronal progenitor cells, using a Nestin-Cre transgene, resulted in severe defects in postnatal eye development, including progressive eye closure, lens rupture, and retinal malformation. These phenotypes were not observed in the presence of a single wild-type allele of either Crk or CrkL. We found that the lens in knockout mice started to rupture and disintegrate between postnatal days 7 and 12, although the structure of the retina was relatively well maintained. As the lens deteriorated further, the outer nuclear layer in the posterior of the retina enlarged and developed ruffles. Cre recombination occurred in the lens and retina of the knockout mice. Furthermore, the posterior lens capsule of the knockout mouse was thinner at postnatal days 0.5 and 3, suggesting that the defective lens capsule caused rupturing of the lens near the posterior pole. These results indicate that Crk and CrkL play essential overlapping roles in postnatal lens development.

Creatine kinase/α-crystallin interaction functions in cataract development

Creatine kinase (CK) is an energy storage enzyme that plays an important role in energy metabolism. CK/phosphocreatine functions as an energy buffer and links ATP production sites with ATP utilization sites. Several key mutations in the αA-crystallin (cryaa) and αB-crystallin (cryab) genes have been linked with autosomal-dominant, hereditary human cataracts. The cryaa-R49C mutation was identified in a four-generation Caucasian family. We previously identified an increase in the quantity of CK complexed with α-crystallin in the lenses of knock-in mice expressing the cryaa-R49C mutation using proteomic analyses. Increased levels of CK in postnatal cataractous lenses may indicate increased ATP requirements during early cataract development. To gain a further understanding of the relationship between CK and α-crystallin, we investigated whether α-crystallin interacts with and forms complexes with CK, in vitro. Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 α-crystallin subunits, with a Kd of 3.3 × 10−7 M, and that the interaction between α-crystallin and CK was endothermic, thermodynamically favorable, and entropy-driven. High-salt concentrations did not affect the interaction between CK and α-crystallin, suggesting that the interaction between CK and α-crystallin is primarily hydrophobic. Gel permeation chromatography (GPC) detected water-soluble α-crystallin and CK complexes, as determined by increased light scattering after complex formation. In addition, CK and α-crystallin formed partially-water-insoluble, high-molecular-mass complexes. Enzyme-linked immunosorbent assay (ELISA)-based enzymatic activity analyses of lens homogenates showed a 17-fold increase in CK activity in the postnatal lenses of cryaa-R49C knock-in mice. These studies indicate that the interaction between α-crystallin and CK is functionally important and that increased CK levels may be necessary to meet the increased ATP demands of ATP-dependent functions in cataractous lenses.

Downregulation of heat shock factor 4 transcription activity via MAPKinase phosphorylation at Serine 299.

Dysfunction of HSF4 is associated with congenital cataracts. HSF4 transcription activity is turned on and regulated by phosphorylation during early postnatal lens development. Our previous data suggested that mutation HSF4b/S299A can upregulate HSF4 transcription activity in vitro, but the biological significance of posttranslational modification on HSF4/S299 during lens development remains unclear. Here, we found that the mutation HSF4/S299A can upregulate the expression of HSP25 and alpha B-crystallin at both protein and mRNA levels in mouse the lens epithelial cell line, but HSF4/S299D does not. Using the rabbit polyclonal antibody against phospho-S299 of HSF4, we found that EGF and ectopic expression of MEK1 can increase the phosphorylation of HSF4/S299 and induce HSF4 sumoylation, and these effects are inhibited by U0126. ERK1/2 can phosphorylate the S299 in HSF4/wt but not in HSF4/S299A in the in vitro kinase assay. Functionally, ectopic MEK1 can inhibit HSF4-controled alpha B-crystallin expression but has less effect on HSF4/S299A. EGF can upregulate phospho-HSF4/S299 and downregulate alpha B-crystallin expression in P3 mouse lens, and this downregulation is suppressed by U0126. During mouse lens development, phosphorylation of HSF4/S299 is downregulated in P3 lens and upregulated in P7 and P14 lens. However, in 2 months old lens, both phosphorylation of HSF4/S299 and total HSF4 protein are decreased. Interestingly, ERK1/2 activity is lower in P3 lens than in P7 and P14 lens, which is in line with the phosphorylation of HSF4/S299. Taken together, our data demonstrate that HSF4/299 is a phosphorylation target of MEK1-ERK1/2, and phosphorylation of S299 is responsible for tuning down HSF4 transcription activity during postnatal lens development.

Spatiotemporal dynamics of canonical Wnt signaling during embryonic eye development and posterior capsular opacification (PCO)

The appropriate spatial and temporal regulation of canonical Wnt signaling is vital for eye development. However, the literature often conflicts on the distribution of canonical Wnt signaling in the eye. Here, using a sensitive mouse transgenic reporter line, we report a detailed re-evaluation of the spatiotemporal dynamics of canonical Wnt signaling in the developing eye. Canonical Wnt activity was dynamic in the optic vesicle and later in the retina, while it was absent from the ectodermal precursors of the lens and corneal epithelium. However, later in corneal development, canonical Wnt reporter activity was detected in corneal stroma and endothelium precursors as they form from the neural crest, although this was lost around birth. Interestingly, while no canonical Wnt signaling was detected in the corneal limbus or basal cells at any developmental stage, it was robust in adult corneal wing and squamous epithelial cells. While canonical Wnt reporter activity was also absent from the postnatal lens, upon lens injury intended to model cataract surgery, it upregulated within 12 h in remnant lens epithelial cells, and co-localized with alpha smooth muscle actin in fibrotic lens epithelial cells from 48 h post-surgery onward. This pattern correlated with downregulation of the inhibitor of canonical Wnt signaling, Dkk3. These data demonstrate that canonical Wnt signaling is dynamic within the developing eye and upregulates in lens epithelial cells in response to lens injury. As canonical Wnt signaling can collaborate with TGFβ to drive fibrosis in other systems, these data offer the first evidence in a lens-injury model that canonical Wnt may synergize with TGFβ signaling to drive fibrotic posterior capsular opacification (PCO).

Regenerating Eye Tissues to Preserve and Restore Vision.

(Cell Stem Cell 22, 834–849; June 1, 2018) The original labeling in Figure 1 incorrectly indicated that the regenerative cells in the postnatal lens epithelium are solely located in the equatorial region. Lineage studies indicate that these regenerative lens epithelial cells, identified in the text as stem/progenitor cells, are located widely in the anterior epithelium but preferentially proliferate in the equatorial region. The labeling has been changed to reflect what is known about the predominant location of this dividing population. We apologize for any confusion caused to our colleagues in the community. Regenerating Eye Tissues to Preserve and Restore VisionStern et al.Cell Stem CellJune 01, 2018In BriefStern et al. review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing. Full-Text PDF Open Archive

Identification of long non-coding RNA and mRNA expression in βΒ2-crystallin knockout mice.

βΒ2-crystallin (CRYBB2) is expressed at an increased level in the postnatal lens cortex and is associated with cataracts. Improved understanding of the underlying biology of cataracts is likely to be critical for the development of early detection strategies and new therapeutics. The present study aimed to identify long non-coding RNAs (lncRNAs) and mRNAs associated with CRYBB2 knockdown (KO)-induced cataracts. RNAs from 3 non-treated mice and 3 CRYBB2 KO mice were analyzed using the Affymetrix GeneChip Mouse Gene 2.0 ST array. A total of 149 lncRNAs and 803 mRNAs were identified to have upregulated expression, including Snora73b, Klk1b22 and Rnu3a, while the expression levels of 180 lncRNAs and 732 mRNAs were downregulated in CRYBB2 KO mice, including Snord82, Snhg9 and Foxn3. This lncRNA and mRNA expression profile of mice with CRYBB2 KO provides a basis for studying the genetic mechanisms of cataract progression.

Transcriptome analysis of developing lens reveals abundance of novel transcripts and extensive splicing alterations

Lens development involves a complex and highly orchestrated regulatory program. Here, we investigate the transcriptomic alterations and splicing events during mouse lens formation using RNA-seq data from multiple developmental stages, and construct a molecular portrait of known and novel transcripts. We show that the extent of novelty of expressed transcripts decreases significantly in post-natal lens compared to embryonic stages. Characterization of novel transcripts into partially novel transcripts (PNTs) and completely novel transcripts (CNTs) (novelty score ≥ 70%) revealed that the PNTs are both highly conserved across vertebrates and highly expressed across multiple stages. Functional analysis of PNTs revealed their widespread role in lens developmental processes while hundreds of CNTs were found to be widely expressed and predicted to encode for proteins. We verified the expression of four CNTs across stages. Examination of splice isoforms revealed skipped exon and retained intron to be the most abundant alternative splicing events during lens development. We validated by RT-PCR and Sanger sequencing, the predicted splice isoforms of several genes Banf1, Cdk4, Cryaa, Eif4g2, Pax6, and Rbm5. Finally, we present a splicing browser Eye Splicer (http://www.iupui.edu/~sysbio/eye-splicer/), to facilitate exploration of developmentally altered splicing events and to improve understanding of post-transcriptional regulatory networks during mouse lens development.

The role of the Shh signaling pathway in radio‐induced cataractogenesis

SummaryBesides the well‐known role in cancer, Shh signaling pathway direct cell proliferation, cell fate determination and EMT, remaining largely expressed in the stem cell compartment of adult tissues. Recently, we proposed Ptch1+/‐ mice as a relevant radiation‐induced cataract mouse model. These mice, develop cataract with a very high incidence (45.2%) and short latency after irradiation with 3 Gy of x‐rays, when radiation is delivered during the early stage of postnatal lens development. Molecular analyses suggest that Shh and TGF‐ß signaling cooperate to promote Ptch1‐associated cataract development by activating EMT and converging on Nanog. Furthermore, using a range of decreasing radiation doses to assess the cataract response in these mice, nonlinear biological responses were observed using doses ≤ 1 Gy, with a suggestion for borderline effects at 2 Gy, both for gross cataract development and for activation of the genetic pathways involved in the molecular pathogenesis of Shh‐associated cataract.These findings highlight a novel function of Shh signaling unrelated to cancer and provide a new animal model to investigate the molecular pathogenesis of cataract formation.

Heat shock factor 4 regulates lens epithelial cell homeostasis by working with lysosome and anti-apoptosis pathways

Activation of Heat shock factor 4-mediated heat shock response is closely associated with postnatal lens development. HSF4 controls the expression of small heat shock proteins (e.g. HSP25 and CRYAB) in lens epithelial cells. However, their roles in modulating lens epithelium homeostasis remain unclear. In this paper, we find that HSF4 is developmentally expressed in mouse lens epithelium and fiber tissue. HSF4 and alpha B-crystallin can selectively protect lens epithelial cells from cisplatin and H2O2 induced apoptosis by stabilizing mitochondrial membrane potential (ΔYm) and reducing ROS production. In addition, to our surprise, HSF4 is involved in upregulating lysosome activity. We found mLEC/HA-Hsf4 cells to have increased DLAD expression, lysosome acidity, cathepsin B activity, and degradation of plasmid DNA and GFP-LC3 protein when compared to mLEC/Hsf4-/- cells. Knocking down Cryab from mLEC/HA-Hsf4 cells inhibits HSF4-mediated lysosome acidification, while overexpression of CRYAB can upregulate cathepsin B activity in mLEC/Hsf4-/- cells. CRAYAB can interact with ATP6V1/A the A subunit of the H+ pump vacuolar ATPase, and is colocalized to lamp1 and lamp2 in the lysosome. Collectively, these results suggest that in addition to modulating anti-apoptosis, HSF4 is able to regulate lysosome activity by at least controlling alpha B-crystallin expression, shedding light on a novel molecular mechanism of HSF4 in regulating lens epithelial cell homeostasis.

The Phosphoinosotide 3-Kinase Catalytic Subunit p110α is Required for Normal Lens Growth.

PurposeSignal transduction pathways influence lens growth, but little is known about the role(s) of the class 1A phosphoinositide 3-kinases (PI3Ks). To further investigate how signaling regulates lens growth, we generated and characterized mice in which the p110α and p110β catalytic subunits of PI3K were conditionally deleted in the mouse lens.MethodsFloxed alleles of the catalytic subunits of PI3K were conditionally deleted in the lens by using MLR10-cre transgenic mice. Lenses of age-matched animals were dissected and photographed. Postnatal lenses were fixed, paraffin embedded, sectioned, and stained with hematoxylin-eosin. Cell proliferation was quantified by labeling S-phase cells in intact lenses with 5-ethynyl-2′-deoxyuridine. Protein kinase B (AKT) activation was examined by Western blotting.ResultsLens-specific deletion of p110α resulted in a significant reduction of eye and lens size, without compromising lens clarity. Conditional knockout of p110β had no effect on lens size or clarity, and deletion of both the p110α and p110β subunits resulted in a phenotype that resembled the p110α single-knockout phenotype. Levels of activated AKT were decreased more in p110α- than in p110β-deficient lenses. A significant reduction in proliferating cells in the germinative zone was observed on postnatal day 0 in p110α knockout mice, which was temporally correlated with decreased lens volume.ConclusionsThese data suggest that the class 1A PI3K signaling pathway plays an important role in the regulation of lens size by influencing the extent and spatial location of cell proliferation in the perinatal period.

Deficiency of the RNA binding protein caprin2 causes lens defects and features of Peters anomaly.

It was recently demonstrated that deficiency of a conserved RNA binding protein (RBP) and RNA granule (RG) component Tdrd7 causes ocular defects including cataracts in human, mouse and chicken, indicating the importance of posttranscriptional regulation in eye development. Here we investigated the function of a second conserved RBP/RG component Caprin2 that is identified by the eye gene discovery tool iSyTE. In situ hybridization, Western blotting and immunostaining confirmed highly enriched expression of Caprin2 mRNA and protein in mouse embryonic and postnatal lens. To gain insight into its function, lens-specific Caprin2 conditional knockout (cKO) mouse mutants were generated using a lens-Cre deleter line Pax6GFPCre. Phenotypic analysis of Caprin2(cKO/cKO) mutants revealed distinct eye defects at variable penetrance. Wheat germ agglutinin staining and scanning electron microscopy demonstrated that Caprin2(cKO/cKO) mutants have an abnormally compact lens nucleus, which is the core of the lens comprised of centrally located terminally differentiated fiber cells. Additionally, Caprin2(cKO/cKO) mutants also exhibited at 8% penetrance a developmental defect that resembles a human condition called Peters anomaly, wherein the lens and the cornea remain attached by a persistent stalk. These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development.

Regulation of Hsf4b nuclear translocation and transcription activity by phosphorylation at threonine 472 (739.2)

Hsf4b, a key regulator of postnatal lens development, is subjected to posttranslational modifications including phosphorylation. However, the phosphorylation sites in Hsf4b and their biological effects on the transcription activity of Hsf4b are poorly understood. Here we examined 17 potential phosphorylation residues in Hsf4b with alanine‐scanning assays and found that a T472A mutation diminished Hsf4b‐mediated expression of Hsp25 and αB‐crystallin. In contrast, the phosphomimetic mutation of T472D enhanced their expression. Further investigation demonstrated that Hsf4b could interact with nuclear‐transporter importin β‐1 and Hsc70 via amino acids 246‐320 and 320‐493, respectively. T472A mutation reduced Hsf4b’s interaction with importin β‐1, while enhancing its interaction with Hsc70, resulting in Hsf4b cytosolic re‐localization, protein instability and transcription activity attenuation. At the upstream, MEK6 was found to interact with Hsf4b and enhance Hsf4b’s nuclear translocation and transcription activity, probably by phosphorylation at sites such as T472. Taken together, our results suggest that phosphorylation of Hsf4b at T472 by protein kinases such as MEK6 regulates Hsf4b interaction with the importin β‐1‐Hsc70 complex, resulting in blockade of it nuclear translocation and transcriptional activity of Hsf4b.Grant Funding Source: national nature sciences foundation of china

Regulation of Hsf4b nuclear translocation and transcription activity by phosphorylation at threonine 472

Hsf4b, a key regulator of postnatal lens development, is subjected to posttranslational modifications including phosphorylation. However, the phosphorylation sites in Hsf4b and their biological effects on the transcription activity of Hsf4b are poorly understood. Here we examined 17 potential phosphorylation residues in Hsf4b with alanine-scanning assays and found that a T472A mutation diminished Hsf4b-mediated expression of Hsp25 and αB-crystallin. In contrast, the phosphomimetic mutation of T472D enhanced their expression. Further investigation demonstrated that Hsf4b could interact with nuclear-transporter importin β-1 and Hsc70 via amino acids 246–320 and 320–493, respectively. T472A mutation reduced Hsf4b's interaction with importin β-1, while enhancing its interaction with Hsc70, resulting in Hsf4b cytosolic re-localization, protein instability and transcription activity attenuation. At the upstream, MEK6 was found to interact with Hsf4b and enhance Hsf4b's nuclear translocation and transcription activity, probably by phosphorylation at sites such as T472. Taken together, our results suggest that phosphorylation of Hsf4b at T472 by protein kinases such as MEK6 regulates Hsf4b interaction with the importin β-1-Hsc70 complex, resulting in blockade of its nuclear translocation and transcriptional activity of Hsf4b.

Bridging integrator protein 3 (Bin3) deletion causes cataract due to morphological abnormalities in lens cells

The lens is a complex epithelial tissue whose highly organized structure is required for its transparency. The BAR superfamily of peripheral membrane proteins control membrane curvature in many contexts and deletion of the BAR family member, Bin3, from mice results in cataracts (a clouding of the lens) associated with actin defects. However, the role that Bin3 plays in postnatal lens development and homeostasis is not known.Hematoxylin and Eosin staining of Bin3 null lenses revealed minor defects in fiber cell packing from birth to 3 weeks postnatal. The defects in fiber cell packing became progressively worse from 1 month until the fiber cells exhibited both vacuoles and disrupted fiber cell packing by 2 months. Scanning electron micrographs show that the elaborate membrane protrusions found on normal lens fiber cells becomes disorganized in Bin3 null lenses. The observed actin defects along with the morphological defect lead us to hypothesize that Bin3 is important for actin nucleation along lens fiber cell lateral membranes. Multiple proteins such as Cdc42, N‐WASP, and ARP2/3 control actin nucleation. In yeast Bin3 recruits Cdc42 to the cell division site and facilitates its activation, thus changing the cytoskeleton. It has not been shown whether this process is conserved in mammalian cells. Current studies suggest that Cdc42 plays a role in changing the cytoskeleton in Bin3 null lenses.Funded by NEI EY12221.Grant Funding Source: NEI EY 12221

The transcription activity of heat shock factor 4b is regulated by FGF2

Heat shock factor 4b has been found to be closely associated with postnatal lens development. It expresses in postnatal lens epithelial and secondary fiber cells and controls the expression of small heat shock proteins which are important for lens homeostasis. However, the signal pathways underlying Hsf4b are still not completely understood. Here we present that Hsf4b transcription activity is regulated by FGF2 a key growth factor that is involved in regulating lens development at multiple stages. FGF2 can promote Hsf4b nuclear-translocation and the expression of Hsp25 and αB-crystallin, the key downstream targets of Hsf4b in the Hsf4b-reconstituted mouse hsf4−/− lens epithelial cells. Further study indicates that FGF2 can induce Hsf4b protein stabilization through ERK1/2-mediated posttranslational phosphorylation or sumoylation. Hsf4b can promote FGF2-induced morphology transition from lens epithelial cell to the fiber cell, and this morphology transition can be inhibited by ERK1/2 inhibitor U0126. Taken together, our data demonstrate that Hsf4b is a novel downstream transcription factor of FGF2, and its transcription activity is associated with FGF2-modulated lens epithelial cell–fiber cell transition.

Developmental Truncations of Connexin 50 by Caspases Adaptively Regulate Gap Junctions/Hemichannels and Protect Lens Cells against Ultraviolet Radiation

The gap junction-forming connexin (Cx) 50 is truncated gradually during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have shown previously that Cx50 is likely to be cleaved by caspase-3 like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry, we mapped two cleavage sites at the C terminus of Cx50 after Glu-368 and Asp-379 and identified caspase-3 and caspase-1 as the responsible proteases, respectively. The activity of caspase-1, like caspase-3, was detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50 in the core region of the lens. The truncated Cx50 fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared with full-length Cx50, caspase-truncated Cx50 showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50 fragments increased cell viability against UV radiation as compared with full-length Cx50. Together, these results suggest that both caspase-1 and -3 are responsible for the cleavage at the C terminus of Cx50 during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50 are likely to adaptively protect cells against elevated oxidative stress associated with lens aging.

The tumor suppressor geneTrp53protects the mouse lens against posterior subcapsular cataracts and the BMP receptor Acvr1 acts as a tumor suppressor in the lens

SUMMARYWe previously found that lenses lacking the Acvr1 gene, which encodes a bone morphogenetic protein (BMP) receptor, had abnormal proliferation and cell death in epithelial and cortical fiber cells. We tested whether the tumor suppressor protein p53 (encoded by Trp53) affected this phenotype. Acvr1 conditional knockout (Acvr1CKO) mouse fiber cells had increased numbers of nuclei that stained for p53 phosphorylated on serine 15, an indicator of p53 stabilization and activation. Deletion of Trp53 rescued the Acvr1CKO cell death phenotype in embryos and reduced Acvr1-dependent apoptosis in postnatal lenses. However, deletion of Trp53 alone increased the number of fiber cells that failed to withdraw from the cell cycle. Trp53CKO and Acvr1;Trp53DCKO (double conditional knockout), but not Acvr1CKO, lenses developed abnormal collections of cells at the posterior of the lens that resembled posterior subcapsular cataracts. Cells from human posterior subcapsular cataracts had morphological and molecular characteristics similar to the cells at the posterior of mouse lenses lacking Trp53. In Trp53CKO lenses, cells in the posterior plaques did not proliferate but, in Acvr1;Trp53DCKO lenses, many cells in the posterior plaques continued to proliferate, eventually forming vascularized tumor-like masses at the posterior of the lens. We conclude that p53 protects the lens against posterior subcapsular cataract formation by suppressing the proliferation of fiber cells and promoting the death of any fiber cells that enter the cell cycle. Acvr1 acts as a tumor suppressor in the lens. Enhancing p53 function in the lens could contribute to the prevention of steroid- and radiation-induced posterior subcapsular cataracts.

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.