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

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.

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Bmp signaling is required for development of primary lens fiber cells

Development ◽

10.1242/dev.129.15.3727 ◽

2002 ◽

Vol 129 (15) ◽

pp. 3727-3737 ◽

Cited By ~ 5

Author(s):

Sonya C. Faber ◽

Michael L. Robinson ◽

Helen P. Makarenkova ◽

Richard A. Lang

Keyword(s):

Cell Differentiation ◽

Mouse Genome Informatics ◽

Bmp Signaling ◽

Dominant Negative ◽

Fiber Cell ◽

Pax6 Gene ◽

Lens Fiber ◽

Lens Fiber Cell ◽

Fiber Cells ◽

Lens Vesicle

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 αA-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.

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Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation

Development ◽

10.1242/dev.129.16.3795 ◽

2002 ◽

Vol 129 (16) ◽

pp. 3795-3802 ◽

Cited By ~ 2

Author(s):

Teri Louise Belecky-Adams ◽

Ruben Adler ◽

David C. Beebe

Keyword(s):

Cell Differentiation ◽

Growth Factor ◽

Cell Elongation ◽

Chicken Embryo ◽

Vitreous Humor ◽

Fiber Cell ◽

Lens Fiber ◽

Lens Fiber Cell ◽

Fiber Cells ◽

Bmp Receptors

Previous studies showed that the retina produces factors that promote the differentiation of lens fiber cells, and identified members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as potential fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the presence of BMP receptors in chicken embryo lenses. We have now observed that phosphorylated SMAD1, an indicator of signaling through BMP receptors, localizes to the nuclei of elongating lens fiber cells. Transduction of chicken embryo retinas and/or lenses with constructs expressing noggin, a secreted protein that binds BMPs and prevents their interactions with their receptors, delayed lens fiber cell elongation and increased cell death in the lens epithelium. In an in vitro explant system, in which chicken embryo or adult bovine vitreous humor stimulates chicken embryo lens epithelial cells to elongate into fiber-like cells, these effects were inhibited by noggin-containing conditioned medium, or by recombinant noggin. BMP2, 4, or 7 were able to reverse the inhibition caused by noggin. Lens cell elongation in epithelial explants was stimulated by treatment with FGF1 or FGF2, alone or in combination with BMP2, but not to the same extent as vitreous humor. These data indicate that BMPs participate in the differentiation of lens fiber cells, along with at least one additional, and still unknown factor.

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Dual function of TGFβ in lens epithelial cell fate: implications for secondary cataract

Molecular Biology of the Cell ◽

10.1091/mbc.e16-12-0865 ◽

2017 ◽

Vol 28 (7) ◽

pp. 907-921 ◽

Cited By ~ 20

Author(s):

Bruce A. Boswell ◽

Anna Korol ◽

Judith A. West-Mays ◽

Linda S. Musil

Keyword(s):

Cell Differentiation ◽

Cell Fate ◽

Lens Epithelial Cell ◽

P38 Map Kinase ◽

Fiber Cell ◽

Dual Function ◽

Lens Fiber ◽

Cell Fates ◽

Secondary Cataract ◽

Fiber Cells

The most common vision-disrupting complication of cataract surgery is posterior capsule opacification (PCO; secondary cataract). PCO is caused by residual lens cells undergoing one of two very different cell fates: either transdifferentiating into myofibroblasts or maturing into lens fiber cells. Although TGFβ has been strongly implicated in lens cell fibrosis, the factors responsible for the latter process have not been identified. We show here for the first time that TGFβ can induce purified primary lens epithelial cells within the same culture to undergo differentiation into either lens fiber cells or myofibroblasts. Marker analysis confirmed that the two cell phenotypes were mutually exclusive. Blocking the p38 kinase pathway, either with direct inhibitors of the p38 MAP kinase or a small-molecule therapeutic that also inhibits the activation of p38, prevented TGFβ from inducing epithelial–myofibroblast transition and cell migration but did not prevent fiber cell differentiation. Rapamycin had the converse effect, linking MTOR signaling to induction of fiber cell differentiation by TGFβ. In addition to providing novel potential therapeutic strategies for PCO, our findings extend the so-called TGFβ paradox, in which TGFβ can induce two disparate cell fates, to a new epithelial disease state.

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Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation

Developmental Biology ◽

10.1016/j.ydbio.2008.03.028 ◽

2008 ◽

Vol 318 (2) ◽

pp. 276-288 ◽

Cited By ~ 96

Author(s):

Haotian Zhao ◽

Tianyu Yang ◽

Bhavani P. Madakashira ◽

Cornelius A. Thiels ◽

Chad A. Bechtle ◽

...

Keyword(s):

Cell Differentiation ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Fibroblast Growth Factor Receptor ◽

Growth Factor Receptor ◽

Fiber Cell ◽

Fibroblast Growth ◽

Lens Fiber ◽

Lens Fiber Cell ◽

Growth Factor Receptor Signaling

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The distribution of the fiber cell intrinsic membrane proteins MP20 and connexin46 in the bovine lens

Journal of Cell Science ◽

10.1242/jcs.103.1.245 ◽

1992 ◽

Vol 103 (1) ◽

pp. 245-257 ◽

Cited By ~ 1

Author(s):

E. Tenbroek ◽

M. Arneson ◽

L. Jarvis ◽

C. Louis

Keyword(s):

Monoclonal Antibody ◽

Fiber Cell ◽

Immunogold Electron Microscopy ◽

Lens Fiber ◽

Lens Fiber Cell ◽

Western Blots ◽

Fiber Cells ◽

Bovine Lens ◽

Narrow Side ◽

Lens Membranes

MP20 is an intrinsic membrane protein previously identified in mammalian lens fiber cells. To identify a possible role for this protein in the lens, the distribution of MP20 and connexin46 has now been examined. Western immunoblotting with an anti-peptide antibody generated to the C-terminal 8 amino acids of MP20 confirmed the presence of this protein in the lens of several different mammalian species. A monoclonal antibody 5H1 was prepared that, in Western blots of bovine lesn membranes, recognized the same component as an antibody to rat connexin46 (Cx46). The apparent molecular mass of this component decreased from 59 kDa to 55 kDa following treatment of lens membranes with alkaline phosphatase. A monoclonal antibody to connexin-related MP70 recognized a 70 kDa component in bovine lens membranes confirming the presence of these two different connexin proteins in bovine lens membranes. To localize MP20 and Cx46 in the bovine lens membrane, lens fiber cell bundles were immunofluorescently labeled with both the MP20 antibody, and the monoclonal antibody to Cx46. Cx46 was identified in large plaques on the broad faces of the lens fiber cells throughout the outer 1 mm of the lens cortex. MP20 colocalized with Cx46 only in a restricted area 0.5 mm to 1.0 mm into the lens. In other regions of the lens, MP20 appeared more diffusely distributed over the fiber cell surface, although apparently concentrated in the ball-and-socket regions at the corners of the narrow side of the inner cortical lens fiber cells. These inner cortical regions were devoid of Cx46. A difference in distribution of these two proteins was confirmed in studies of immunofluorescently labeled lens cryosections. Furthermore, immunogold electron microscopy of purified lens membranes identified MP20 in both junctional regions (with Cx46) and in single membranes. These results provide evidence for a role for MP20 in mammalian lens fiber cell junctional formation or organization.

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The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology

Human Molecular Genetics ◽

10.1093/hmg/ddaa096 ◽

2020 ◽

Vol 29 (12) ◽

pp. 2076-2097 ◽

Cited By ~ 2

Author(s):

Carrie E Barnum ◽

Salma Al Saai ◽

Shaili D Patel ◽

Catherine Cheng ◽

Deepti Anand ◽

...

Keyword(s):

Single Molecule ◽

Cell Morphology ◽

Fiber Cell ◽

Maturation Stage ◽

Lens Fiber ◽

Protein Fluorescence ◽

Pediatric Cataract ◽

Fiber Cells ◽

Ribonucleoprotein Complexes ◽

Nuclear Degradation

Abstract Mutations of the RNA granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7−/− mice. Early postnatal Tdrd7−/− animals precipitously develop cataract suggesting a global-level breakdown/misregulation of key cellular processes. High-throughput RNA sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal modulator, HSPB1, among high-priority downregulated candidates in Tdrd7−/− lens. A protein fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE)-coupled mass spectrometry screen also identified HSPB1 downregulation, offering independent support for its importance to Tdrd7−/− cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance, and its reduction in Tdrd7−/− lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7−/− cataract. In agreement, scanning electron microscopy (SEM) revealed abnormal fiber cell morphology in Tdrd7−/− lenses. Further, abnormal phalloidin and wheat germ agglutinin (WGA) staining of Tdrd7−/− fiber cells, particularly those exhibiting nuclear degradation, reveals distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, RNA immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in differentiating fiber cells, suggesting that TDRD7–ribonucleoprotein complexes may be involved in optimal buildup of key factors. Finally, Hspb1 knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7’s novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.

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