Elevated tropomyosin expression is associated with epithelial-mesenchymal transition of lens epithelial cells (original) (raw)
Related papers
Purpose: Epithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsular cataracts. Central to EMT is the formation of actin stress fibers. Targeting specific, stress fiber associated tropomyosin in epithelial cells may be a means to prevent stress fiber formation and repress lens EMT. Methods: We identified Tpm isoforms in mouse immortalized lens epithelial cells and isolated whole lenses by semi-quantitative PCR followed Sanger sequencing. We focused on the role of one particular tropomyosin isoform, Tpm3.1, in EMT. To stimulate EMT, we cultured cells or native lenses in TGFβ. To test the function of Tpm3.1, we exposed cells or whole lenses to a Tpm3.1-specific chemical inhibitor, TR100, as well as investigated lenses from Tpm3.1 knockout mice. We examined stress fiber formation by confocal microscopy and assessed EMT progression by αsma mRNA (qPCR) and protein (WES immunoassay) analysis. Results: Lens epithelial cells express eight tropomyosin isoforms...
International Journal of Molecular Sciences
Fibrotic cataracts have been attributed to transforming growth factor-beta (TGF-β)-induced epithelial-to-mesenchymal transition (EMT). Using mouse knockout (KO) models, our laboratory has identified MMP9 as a crucial protein in the TGF-β-induced EMT process. In this study, we further revealed an absence of alpha-smooth muscle actin (αSMA) and filamentous-actin (F-actin) stress fibers in MMP9KO mouse lens epithelial cell explants (LECs). Expression analysis using NanoString revealed no marked differences in αSMA (ACTA2) and beta-actin (β-actin) (ACTB) mRNA between the lenses of TGF-β-overexpressing (TGF-βtg) mice and TGF-βtg mice on a MMP9KO background. We subsequently conducted a protein array that revealed differential regulation of proteins known to be involved in actin polymerization and cell migration in TGF-β-treated MMP9KO mouse LECs when compared to untreated controls. Immunofluorescence analyses using rat LECs and the novel MMP9-specific inhibitor, JNJ0966, revealed similar ...
Experimental Eye Research, 2009
Transforming growth factor beta (TGFβ) has been known to play a role in anterior subcapsular cataract (ASC) formation and posterior capsule opacification (PCO), both of which are fibrotic pathologies of the lens. Several models have been utilized to study ASC formation, including the TGFβ1 transgenic mouse model and the ex-vivo rat lens model. A distinct characteristic of ASC development within these models includes the formation of isolated fibrotic plaques or opacities which form beneath the lens capsule. A hallmark feature of ASC formation is the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) into myofibroblasts. Recently, the matrix metalloproteinases (MMPs) have been implicated in the formation of these cataracts through their involvement in EMT. In the present study, we sought to further investigate the role of MMPs in subcapsular cataract formation in a time course manner, through the examination of gene expression and morphological changes which occur during this process. RT-QPCR and immunohistochemical analysis was carried out on lenses treated with TGFβ for a period of 2, 4 and 6 days. Laser capture microdissection (LCM) was utilized to specifically isolate cells within the plaque region and cells from the adjacent epithelium in lenses treated for a 6 day period. Multilayering of LECs was observed as early as day 2, which preceded the presence of alpha smooth muscle actin (α-SMA) immunoreactivity that was evident following 4 days of treatment with TGFβ. A slight reduction in E-cadherin mRNA was detected at day 2, although this was not significant until the day 4 time point. Importantly, our results also indicate an early induction of MMP-9 mRNA following 2 days of TGFβ treatment, whereas MMP-2 was found to be upregulated at the later 4 day time point. Further experiments using FHL 124 cells show an induction in MMP-2 protein levels following treatment with recombinant MMP-9. Together these findings suggest an upstream role for MMP-9 in ASC formation.
Frontiers in Cell and Developmental Biology
Injury to the ocular lens perturbs cell-cell and cell-capsule/basement membrane interactions leading to a myriad of interconnected signaling events. These events include cell-adhesion and growth factor-mediated signaling pathways that can ultimately result in the induction and progression of epithelial-mesenchymal transition (EMT) of lens epithelial cells and fibrosis. Since the lens is avascular, consisting of a single layer of epithelial cells on its anterior surface and encased in a matrix rich capsule, it is one of the most simple and desired systems to investigate injury-induced signaling pathways that contribute to EMT and fibrosis. In this review, we will discuss the role of key cell-adhesion and mechanotransduction related signaling pathways that regulate EMT and fibrosis in the lens.
Investigative Opthalmology & Visual Science
PURPOSE. Transforming growth factor-b-induced epithelial-mesenchymal transition (EMT) is one of the main causes of posterior capsular opacification (PCO) or secondary cataract; however, the signaling events involved in TGF-b-induced PCO have not been fully characterized. Here, we focus on examining the role of b-catenin/cyclic AMP response element-binding protein (CREB)-binding protein (CBP) and b-catenin/T-cell factor (TCF)dependent signaling in regulating cytoskeletal dynamics during TGF-b-induced EMT in lens epithelial explants. METHODS. Rat lens epithelial explants were cultured in medium M199 in the absence of serum. Explants were treated with TGF-b2 in the presence or absence of the b-catenin/CBP interaction inhibitor, ICG-001, or the b-catenin/TCF interaction inhibitor, PNU-74654. Western blot and immunofluorescence experiments were carried out and analyzed. RESULTS. An increase in the expression of fascin, an actin-bundling protein, was observed in the lens explants upon stimulation with TGF-b, and colocalized with F-actin filaments. Inhibition of b-catenin/CBP interactions, but not b-catenin/TCF interactions, led to a decrease in TGF-b-induced fascin and stress fiber formation, as well as a decrease in the expression of known markers of EMT, a-smooth muscle actin (a-SMA) and matrix metalloproteinase 9 (MMP9). In addition, inhibition of b-catenin/CBP-dependent signaling also prevented TGF-binduced downregulation of epithelial cadherin (E-cadherin) in lens explants. CONCLUSIONS. We show that b-catenin/CBP-dependent signaling regulates fascin, MMP9, and a-SMA expression during TGF-b-induced EMT. We demonstrate that b-catenin/CBP-dependent signaling is crucial for TGF-b-induced EMT in the lens.
Biochemical and Biophysical Research Communications, 2008
To understand lens fiber cell elongation-and differentiation-associated cytoskekeletal 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 GTPasetargeted transgenic mouse lenses. These data identify the ERM proteins, which crosslink 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.
PURPOSE: Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a key pathologic mechanism underlying cataract. Two members of the transforming growth factor-β (TGFβ) superfamily, TGFβ and bone morphogenetic protein-7 (BMP-7) have functionally distinct roles in EMT. While TGFβ is a potent inducer of EMT, BMP-7 counteracts the fibrogenic activity of TGFβ. We examine the modulating effect of BMP-7 on TGFβ-induced EMT in LECs. METHODS: Rat lens epithelial explants were treated exogenously with TGFβ2 alone or in combination with BMP-7 for up to 5 days. Expression levels of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), and phosphorylated downstream Smads were determined using immunofluorescence and Western blotting. Reverse transcriptase quantitative PCR (RT-qPCR) was used to study gene expression levels of EMT markers and downstream BMP target genes, including the Inhibitors of differentiation (Id). RESULTS: Transforming growth factor-β2 induced LECs to transdifferentiate into myofibroblastic cells. Addition of BMP-7 suppressed TGFβ2-induced α-SMA protein levels and mesenchymal gene expression, with retention of E-cadherin and β-catenin expression to the cell membrane. Addition of BMP-7 prevented lens capsular wrinkling and cellular loss associated with TGFβ2-induced EMT over the 5-day treatment period. The inhibitory effect of BMP-7 was accompanied by an early induction of pSmad1/5 and suppression of TGFβ2-induced pSmad2/3. Treatment with TGFβ2 alone suppressed gene expression of Id2/3 and addition of BMP-7 restored Id2/3 expression. CONCLUSIONS: Exogenous administration of BMP-7 abrogated TGFβ2-induced EMT in rat lens epithelial explants. Understanding the complex interplay between the TGFβ- and BMP-7-associated Smad signaling pathways and their downstream target genes holds therapeutic promise in cataract prevention.
Investigative Opthalmology & Visual Science
PURPOSE. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a key pathologic mechanism underlying cataract. Two members of the transforming growth factor-b (TGFb) superfamily, TGFb and bone morphogenetic protein-7 (BMP-7) have functionally distinct roles in EMT. While TGFb is a potent inducer of EMT, BMP-7 counteracts the fibrogenic activity of TGFb. We examine the modulating effect of BMP-7 on TGFb-induced EMT in LECs. METHODS. Rat lens epithelial explants were treated exogenously with TGFb2 alone or in combination with BMP-7 for up to 5 days. Expression levels of E-cadherin, b-catenin, a-smooth muscle actin (a-SMA), and phosphorylated downstream Smads were determined using immunofluorescence and Western blotting. Reverse transcriptase quantitative PCR (RT-qPCR) was used to study gene expression levels of EMT markers and downstream BMP target genes, including the Inhibitors of differentiation (Id). RESULTS. Transforming growth factor-b2 induced LECs to transdifferentiate into myofibroblastic cells. Addition of BMP-7 suppressed TGFb2-induced a-SMA protein levels and mesenchymal gene expression, with retention of E-cadherin and b-catenin expression to the cell membrane. Addition of BMP-7 prevented lens capsular wrinkling and cellular loss associated with TGFb2-induced EMT over the 5-day treatment period. The inhibitory effect of BMP-7 was accompanied by an early induction of pSmad1/5 and suppression of TGFb2induced pSmad2/3. Treatment with TGFb2 alone suppressed gene expression of Id2/3 and addition of BMP-7 restored Id2/3 expression. CONCLUSIONS. Exogenous administration of BMP-7 abrogated TGFb2-induced EMT in rat lens epithelial explants. Understanding the complex interplay between the TGFb-and BMP-7associated Smad signaling pathways and their downstream target genes holds therapeutic promise in cataract prevention.
The murine lens: A model to investigate in vivo epithelial-mesenchymal transition
Developmental Dynamics
Epithelial-mesenchymal transition (EMT) produces myofibroblasts that contribute to the formation of fibrotic tissue with an impairment of tissue homeostasis and functionality. The crystalline lens of the eye is a unique transparent and isolated tissue. The lens vesicle becomes isolated from the surface ectoderm, its cells are all contained as they line the inner surface of the lens capsule. Clinically the formation of fibrotic tissue by the lens epithelial cells causes a type of cataract or opacification and contraction of the lens capsule postcataract surgery. Production of EMT in the intact animal lens by using specific gene transfer to the lens or experimental lens injury has been shown to be a powerful tool to investigate EMT processes. It is not easy to uncover whether the origin of the myofibroblast is epithelial cell-derived or from other cell lineages in fibrotic tissues. However, myofibroblasts that appear in the crystalline lens pathology are totally derived from the lens epithelial cells for the reasons mentioned above. Here, we report on different animal models of lens EMT, using either transgenic approaches or injury to study the biological aspects of EMT.
MMP inhibition prevents human lens epithelial cell migration and contraction of the lens capsule
The British journal of ophthalmology, 2004
Purpose: The development of posterior capsule contraction following cataract surgery is caused by the activity of residual lens epithelial cells. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes, which are essential for cell migration and cell mediated contraction following wound healing. The authors investigated whether inhibiting MMP activity can reduce lens epithelial cell migration and as a result, lead to a reduction in cell mediated capsule contraction. Methods: Human donor lens capsules were cultured and treated with a broad spectrum MMP inhibitor, Ilomastat (GM6001). MMP-2 and MMP-9 production were determined by ELISA. Cell migration onto the posterior capsule and capsule contraction were digitally measured. Results: MMP inhibition significantly reduced lens epithelial cell migration onto the posterior capsule (p,0.05), and a reduction in capsule contraction was observed (p,0.05). Conclusions: Ilomastat significantly reduced lens epithelial cell migration onto the posterior capsule surface and inhibited capsule contraction. MMP inhibition may have a role in the therapeutic treatment of posterior capsule opacification.