Influence of the calcium voltage-gated channel auxiliary subunit (CACNA2D1) absence on intraocular pressure in mice.

The etiology of elevated intraocular pressure (IOP), a major risk factor for glaucoma (optic nerve atrophy), is poorly understood despite continued efforts. Although the gene variant of CACNA2D1 (encoding α2δ1), a calcium voltage-gated channel auxiliary subunit, has been reported to be associated with primary open-angle glaucoma, and the pharmacological mitigation of α2δ1 activity by pregabalin lowers IOP, the cellular basis for α2δ1 role in the modulation of IOP remains unclear. Our recent findings reveled readily detectable levels of α2δ1 and its ligand thrombospondin in the cytoskeletome fraction of human trabecular meshwork (TM) cells. To understand the direct role of α2δ1 in the modulation of IOP, we evaluated α2δ1 null mice for changes in IOP and found a moderate (∼10%) but significant decrease in IOP compared to littermate wild type control mice. Additionally, to gain cellular insights into α2δ1 antagonist (pregabalin) induced IOP changes, we assessed pregabalin's effects on human TM cell actin cytoskeletal organization and cell adhesive interactions in comparison with a Rho kinase inhibitor (Y27632), a known ocular hypotensive agent. Unlike Y27632, pregabalin did not have overt effects on cell morphology, actin cytoskeletal organization, or cell adhesion in human TM cells. These results reveal a modest but significant decrease in IOP in α2δ1 deficient mice, and this response appears to be not associated with the contractile and cell adhesive characteristics of TM cells based on the findings of pregabalin effects on isolated TM cells. Therefore, the mechanism by which pregabalin lowers IOP remains elusive.

Influence of Growth Differentiation Factor 15 on Intraocular Pressure in Mice.

Growth differentiation factor 15 (GDF15), a stress-sensitive cytokine, and a distant member of the transforming growth factor ß superfamily, has been shown to exhibit increased levels with aging, and in various age-related pathologies. Although GDF15 levels are elevated in the aqueous humor (AH) of glaucoma (optic nerve atrophy) patients, the possible role of this cytokine in the modulation of intraocular pressure (IOP) or AH outflow is unknown. The current study addresses this question using transgenic mice expressing human GDF15 and GDF15 null mice, and by perfusing enucleated mouse eyes with recombinant human GDF15 (rhGDF15). Treatment of primary cultures of human trabecular meshwork cells with a telomerase inhibitor, an endoplasmic reticulum stress-inducing agent, hydrogen peroxide, or an autophagy inhibitor resulted in significant elevation in GDF15 levels relative to the respective control cells. rhGDF15 stimulated modest but significant increases in the expression of genes encoding the extracellular matrix, cell adhesion proteins, and chemokine receptors (C-C chemokine receptor type 2) in human trabecular meshwork cells compared with controls, as deduced from the differential transcriptional profiles using RNA-sequencing analysis. There was a significant increase in IOP in transgenic mice expressing human GDF15, but not in GDF15 null mice, compared with the respective wild-type control mice. The AH outflow facility was decreased in enucleated wild-type mouse eyes perfused with rhGDF15. Light microcopy-based histologic examination of the conventional AH outflow pathway tissues did not reveal identifiable differences between the GDF15-targeted and control mice. Taken together, these results reveal the modest elevation of IOP in mice expressing human GDF15 possibly stemming from decreased AH outflow through the trabecular pathway.

Glypican-4 regulated actin cytoskeletal reorganization in glucocorticoid treated trabecular meshwork cells and involvement of Wnt/PCP signaling.

A common adverse response to the clinical use of glucocorticoids (GCs) is elevated intraocular pressure (IOP) which is a major risk factor for glaucoma. Elevated IOP arises due to impaired outflow of aqueous humor (AH) through the trabecular meshwork (TM). Although GC-induced changes in actin cytoskeletal dynamics, contractile characteristics, and cell adhesive interactions of TM cells are believed to influence AH outflow and IOP, the molecular mechanisms mediating changes in these cellular characteristics are poorly understood. Our studies focused on evaluating changes in the cytoskeletal and cytoskeletal-associated protein (cytoskeletome) profile of human TM cells treated with dexamethasone (Dex) using label-free mass spectrometric quantification, identified elevated levels of specific proteins known to regulate actin stress fiber formation, contraction, actin networks crosslinking, cell adhesion, and Wnt signaling, including LIMCH1, ArgBP2, CNN3, ITGBL1, CTGF, palladin, FAT1, DIAPH2, EPHA4, SIPA1L1, and GPC4. Several of these proteins colocalized with the actin cytoskeleton and underwent alterations in distribution profile in TM cells treated with Dex, and an inhibitor of Abl/Src kinases. Wnt/Planar Cell Polarity (PCP) signaling agonists-Wnt5a and 5b were detected prominently in the cytoskeletome fraction of TM cells, and studies using siRNA to suppress expression of glypican-4 (GPC4), a known modulator of the Wnt/PCP pathway revealed that GPC4 deficiency impairs Dex induced actin stress fiber formation, and activation of c-Jun N-terminal Kinase (JNK) and Rho kinase. Additionally, while Dex augmented, GPC4 deficiency suppressed the formation of actin stress fibers in TM cells in the presence of Dex and Wnt5a. Taken together, these results identify the GPC4-dependent Wnt/PCP signaling pathway as one of the crucial upstream regulators of Dex induced actin cytoskeletal reorganization and cell adhesion in TM cells, opening an opportunity to target the GPC4/Wnt/PCP pathway for treatment of ocular hypertension in glaucoma.

Glucocorticoids Preferentially Influence Expression of Nucleoskeletal Actin Network and Cell Adhesive Proteins in Human Trabecular Meshwork Cells.

Clinical use of glucocorticoids is associated with increased intraocular pressure (IOP), a major risk factor for glaucoma. Glucocorticoids have been reported to induce changes in actin cytoskeletal organization, cell adhesion, extracellular matrix, fibrogenic activity, and mechanical properties of trabecular meshwork (TM) tissue, which plays a crucial role in aqueous humor dynamics and IOP homeostasis. However, we have a limited understanding of the molecular underpinnings regulating these myriad processes in TM cells. To understand how proteins, including cytoskeletal and cell adhesion proteins that are recognized to shuttle between the cytosolic and nuclear regions, influence gene expression and other cellular activities, we used proteomic analysis to characterize the nuclear protein fraction of dexamethasone (Dex) treated human TM cells. Treatment of human TM cells with Dex for 1, 5, or 7 days led to consistent increases (by ≥ two-fold) in the levels of various actin cytoskeletal regulatory, cell adhesive, and vesicle trafficking proteins. Increases (≥two-fold) were also observed in levels of Wnt signaling regulator (glypican-4), actin-binding chromatin modulator (BRG1) and nuclear actin filament depolymerizing protein (MICAL2; microtubule-associated monooxygenase, calponin and LIM domain containing), together with a decrease in tissue plasminogen activator. These changes were independently further confirmed by immunoblotting analysis. Interestingly, deficiency of BRG1 expression blunted the Dex-induced increases in the levels of some of these proteins in TM cells. In summary, these findings indicate that the widely recognized changes in actin cytoskeletal and cell adhesive attributes of TM cells by glucocorticoids involve actin regulated BRG1 chromatin remodeling, nuclear MICAL2, and glypican-4 regulated Wnt signaling upstream of the serum response factor/myocardin controlled transcriptional activity.

Mechanical Load and Piezo1 Channel Regulated Myosin II Activity in Mouse Lenses.

The cytoarchitecture and tensile characteristics of ocular lenses play a crucial role in maintaining their transparency and deformability, respectively, which are properties required for the light focusing function of ocular lens. Calcium-dependent myosin-II-regulated contractile characteristics and mechanosensitive ion channel activities are presumed to influence lens shape change and clarity. Here, we investigated the effects of load-induced force and the activity of Piezo channels on mouse lens myosin II activity. Expression of the Piezo1 channel was evident in the mouse lens based on immunoblot and immufluorescence analyses and with the use of a Piezo1-tdT transgenic mouse model. Under ex vivo conditions, change in lens shape induced by the load decreased myosin light chain (MLC) phosphorylation. While the activation of Piezo1 by Yoda1 for one hour led to an increase in the levels of phosphorylated MLC, Yoda1 treatment for an extended period led to opacification in association with increased calpain activity and degradation of membrane proteins in ex vivo mouse lenses. In contrast, inhibition of Piezo1 by GsMTx4 decreased MLC phosphorylation but did not affect the lens tensile properties. This exploratory study reveals a role for the mechanical load and Piezo1 channel activity in the regulation of myosin II activity in lens, which could be relevant to lens shape change during accommodation.

Elevated Levels of Growth/Differentiation Factor-15 in the Aqueous Humor and Serum of Glaucoma Patients.

Dysregulated levels of growth/differentiation factor-15 (GDF15), a divergent member of the transforming growth factor-beta super family, have been found to be associated with the pathology of various diseases. In this study, we evaluated the levels of GDF15 in aqueous humor (AH) and serum samples derived from primary open-angle glaucoma (POAG) and age- and gender-matched non-glaucoma (cataract) patients to assess the plausible association between GDF15 and POAG. GDF15 levels were determined using an enzyme-linked immunosorbent assay, and data analysis was performed using the Wilcoxon rank sum test, or the Kruskal-Wallis test and linear regression. GDF15 levels in the AH (n = 105) of POAG patients were significantly elevated (by 7.4-fold) compared to cataract patients (n = 117). Serum samples obtained from a subgroup of POAG patients (n = 41) also showed a significant increase in GDF15 levels (by 50%) compared to cataract patients. GDF15 levels were elevated in male, female, African American, and Caucasian POAG patients. This study reveals a significant and marked elevation of GDF15 levels in the AH of POAG patients compared to non-glaucoma cataract control patients. Although serum GDF15 levels were also elevated in POAG patients, the magnitude of difference was much smaller relative to that found in the AH.

Role of vasorin, an anti-apoptotic, anti-TGF-ß and hypoxia-induced glycoprotein in the trabecular meshwork cells and glaucoma.

Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open-angle glaucoma patients compared to non-glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti-apoptotic and anti-TGF-ß activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF-ß2-induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride-induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF-α-induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF-ß induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma.

Drebrin, an actin-binding protein, is required for lens morphogenesis and growth.

BACKGROUND: Lens morphogenesis, architecture, and clarity are known to be critically dependent on actin cytoskeleton organization and cell adhesive interactions. There is limited knowledge, however regarding the identity and role of key proteins regulating actin cytoskeletal organization in the lens. This study investigated the role of drebrin, a developmentally regulated actin-binding protein, in mouse lens development by generating and characterizing a conditional knockout (cKO) mouse model using the Cre-LoxP recombination approach. RESULTS: Drebrin E, a splice variant of DBN1 is a predominant isoform expressed in the mouse lens and exhibits a maturation-dependent downregulation. Drebrin co-distributes with actin in both epithelium and fibers. Conditional deficiency (both haploinsufficiency and complete absence) of drebrin results in disrupted lens morphogenesis leading to cataract and microphthalmia. The drebrin cKO lens reveals a dramatic decrease in epithelial height and width, E-cadherin, and proliferation, and increased apoptotic cell death and expression of α-smooth muscle actin, together with severely impaired fiber cell organization, polarity, and cell-cell adhesion. CONCLUSIONS: This study demonstrates the requirement of drebrin in lens development and growth, with drebrin deficiency leading to impaired lens morphogenesis and microphthalmia.

Absence of S100A4 in the mouse lens induces an aberrant retina-specific differentiation program and cataract.

S100A4, a member of the S100 family of multifunctional calcium-binding proteins, participates in several physiological and pathological processes. In this study, we demonstrate that S100A4 expression is robustly induced in differentiating fiber cells of the ocular lens and that S100A4 (-/-) knockout mice develop late-onset cortical cataracts. Transcriptome profiling of lenses from S100A4 (-/-) mice revealed a robust increase in the expression of multiple photoreceptor- and Müller glia-specific genes, as well as the olfactory sensory neuron-specific gene, S100A5. This aberrant transcriptional profile is characterized by corresponding increases in the levels of proteins encoded by the aberrantly upregulated genes. Ingenuity pathway network and curated pathway analyses of differentially expressed genes in S100A4 (-/-) lenses identified Crx and Nrl transcription factors as the most significant upstream regulators, and revealed that many of the upregulated genes possess promoters containing a high-density of CpG islands bearing trimethylation marks at histone H3K27 and/or H3K4, respectively. In support of this finding, we further documented that S100A4 (-/-) knockout lenses have altered levels of trimethylated H3K27 and H3K4. Taken together, our findings suggest that S100A4 suppresses the expression of retinal genes during lens differentiation plausibly via a mechanism involving changes in histone methylation.

Global phosphotyrosinylated protein profile of cell-matrix adhesion complexes of trabecular meshwork cells.

Dysregulation of the mechanical properties and cell adhesive interactions of trabecular meshwork (TM) are known to impair aqueous humor drainage and elevate intraocular pressure in glaucoma patients. The identity of regulatory mechanisms underlying TM mechanotransduction, however, remains elusive. Here we analyzed the phosphotyrosine proteome of human TM cell-extracellular matrix (ECM) adhesion complexes, which play a key role in sensing and transducing extracellular chemical and mechanical cues into intracellular activities, using a two-level affinity pull-down (phosphotyrosine antibody and titanium dioxide beads) method and mass spectrometry. This analysis identified ~1,000 tyrosine-phosphorylated proteins of TM cell-ECM adhesion complexes. Many consensus adhesome proteins were found to be tyrosine phosphorylated. Interestingly, several of the phosphotyrosinylated proteins found in TM cell-ECM adhesion complexes are known to be required for podocyte glomerular filtration, indicating the existence of molecular parallels that are likely relevant to the shared fluid barrier and filtration functions of the two mechanosensitive cell types.

Calponin-3 deficiency augments contractile activity, plasticity, fibrogenic response and Yap/Taz transcriptional activation in lens epithelial cells and explants.

The transparent ocular lens plays a crucial role in vision by focusing light on to the retina with loss of lens transparency leading to impairment of vision. While maintenance of epithelial phenotype is recognized to be essential for lens development and function, knowledge of the identity of different molecular mechanisms regulating lens epithelial characteristics remains incomplete. This study reports that CNN-3, the acidic isoform of calponin, an actin binding contractile protein, is expressed preferentially and abundantly relative to the basic and neutral isoforms of calponin in the ocular lens, and distributes predominantly to the epithelium in both mouse and human lenses. Expression and MEKK1-mediated threonine 288 phosphorylation of CNN-3 is induced by extracellular cues including TGF-ß2 and lysophosphatidic acid. Importantly, siRNA-induced deficiency of CNN3 in lens epithelial cell cultures and explants results in actin stress fiber reorganization, stimulation of focal adhesion formation, Yap activation, increases in the levels of α-smooth muscle actin, connective tissue growth factor and fibronectin, and decreases in E-cadherin expression. These results reveal that CNN3 plays a crucial role in regulating lens epithelial contractile activity and provide supporting evidence that CNN-3 deficiency is associated with the induction of epithelial plasticity, fibrogenic activity and mechanosensitive Yap/Taz transcriptional activation.

Ankyrin-G regulated epithelial phenotype is required for mouse lens morphogenesis and growth.

Epithelial cell polarity, adhesion, proliferation, differentiation and survival are essential for morphogenesis of various organs and tissues including the ocular lens. The molecular mechanisms regulating the lens epithelial phenotype however, are not well understood. Here we investigated the role of scaffolding protein ankyrin-G (AnkG) in mouse lens development by conditional suppression of AnkG expression using the Cre-LoxP recombination approach. AnkG, which serves to link integral membrane proteins to the spectrin/actin cytoskeleton, was found to distribute predominantly to the lateral membranes of lens epithelium with several isoforms of the protein being detected in the mouse lens. Conditional deficiency of AnkG impaired mouse lens morphogenesis starting from embryonic stage E15.5, with neonatal (P1) AnkG cKO lenses exhibiting overt abnormalities in shape, size, epithelial cell height, sheet length and lateral membrane assembly together with defective fiber cell orientation relative to lenses from littermate AnkG floxed or Cre expressing mice. Severe disruptions in E-cadherin/ß-catenin-based adherens junctions, and the membrane organization of spectrin-actin cytoskeleton, ZO-1, connexin-50 and Na+-K+-ATPase were noted in AnkG deficient lenses, along with detection in lens epithelium of α-smooth muscle actin, a marker of epithelial to mesenchymal transition. Moreover, lens epithelial cell proliferation and survival were severely compromised while differentiation appears to be normal in AnkG deficient mouse lenses. Collectively, these results indicate that AnkG regulates establishment of the epithelial phenotype via lateral membrane assembly, stabilization of E-cadherin-based cell-cell junctions, polarity and membrane organization of transport and adhesion proteins and the spectrin-actin skeleton, and provide evidence for an obligatory role for AnkG in lens morphogenesis and growth.

Switching of α-Catenin From Epithelial to Neuronal Type During Lens Epithelial Cell Differentiation.

Purpose: Ocular lens fiber cell elongation, differentiation, and compaction are associated with extensive reorganization of cell adhesive interactions and cytoskeleton; however, our knowledge of proteins critical to these events is still evolving. This study characterizes the distribution pattern of neuronal-specific α-catenin (αN-catenin) and its interaction with the N-cadherin-associated adherens junctions (AJs) and their stability in the mouse lens fibers. Methods: Expression and distribution of αN-catenin in developing mouse and adult human lenses was determined by RT-PCR, immunoblot, and immunofluorescence analyses. Characterization of αN-catenin and N-cadherin interacting proteins and colocalization analyses were performed using immunoprecipitation, mass spectrometry, and confocal imaging. Effects of periaxin deficiency on the stability of lens fiber cell AJs were evaluated using perixin-null mice. Results: αN-catenin exhibits discrete distribution to lens fibers in both mouse and human lenses, undergoing a robust up-regulation during fiber cell differentiation and maturation. Epithelial-specific α-catenin (αE-catenin), in contrast, distributes primarily to the lens epithelium. αN-catenin and N-cadherin reciprocally coimmunoprecipitate and colocalize along with ß-catenin, actin, spectrin, vinculin, Armadillo repeat protein deleted in velo-cardio-facial syndrome homolog, periaxin, and ankyrin-B in lens fibers. Fiber cells from periaxin-null mouse lenses revealed disrupted N-cadherin/αN-catenin-based AJs. Conclusions: These results suggest that the discrete shift in α-catenin expression from αE-catenin to αN-catenin subtype that occurs during lens epithelial cell differentiation may play a key role in fiber cell cytoarchitecture by regulating the assembly and stability of N-cadherin-based AJs. This study also provides evidence for the importance of the fiber cell-specific cytoskeletal interacting periaxin, in the stability of N-cadherin/αN-catenin-based AJs in lens fibers.

Vertebrate Lonesome Kinase Regulated Extracellular Matrix Protein Phosphorylation, Cell Shape, and Adhesion in Trabecular Meshwork Cells.

Glaucoma, a leading cause of irreversible blindness, is commonly associated with elevated intraocular pressure (IOP) due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although dysregulated production and organization of extracellular matrix (ECM) is presumed to increase resistance to AH outflow and elevate IOP by altering TM cell contractile and adhesive properties, it is not known whether regulation of ECM protein phosphorylation via the secretory vertebrate lonesome kinase (VLK) influences TM cellular characteristics. Here, we tested this possibility. Experiments carried out in this study reveal that the 32 kDa protein is a prominent VLK isoform detectable in lysates and conditioned media (CM) of human TM cells. Increased levels of VLK were observed in CM of TM cells subjected to cyclic mechanical stretch, or treated with dexamethasone, TGF-ß2, and TM cells expressing constitutively active RhoA GTPase. Downregulation of VLK expression in TM cells using siRNA decreased tyrosine phosphorylation (TyrP) of ECM proteins and focal adhesions, and induced changes in cell shape in association with reduced levels of actin stress fibers and phospho-paxillin. VLK was also demonstrated to regulate TGF-ß2-induced TyrP of ECM proteins. Taken together, these results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins, and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers, and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma. J. Cell. Physiol. 232: 2447-2460, 2017. © 2016 Wiley Periodicals, Inc.

Corneal Endothelial Cell Migration and Proliferation Enhanced by Rho Kinase (ROCK) Inhibitors in In Vitro and In Vivo Models.

Purpose: To explore the role of Rho-associated kinases (ROCK) in corneal physiology and regeneration, and the effects of suppressing its activity in stimulating corneal endothelial cell proliferation and migration in vitro and in vivo. Methods: Immunohistochemistry was performed to detect RhoA and ROCK-1 and ROCK-2 in human corneal tissue. Adult porcine corneal endothelial cells (CECs) were isolated, grown to confluence, and further characterized. Under the treatment of ROCK inhibitors, changes in the cellular distribution profile of ZO-1 and F-actin were examined by immunofluorescence staining. Corneal endothelial cells migration was evaluated by scratch assay and analyzed with Axiovision software. Cell proliferation was quantified using Click-iT EdU HCS Assay. In vivo, the corneal endothelia of rabbits were surgically injured and H-1152 was topically applied for 10 days. Progress of wound healing was evaluated daily by monitoring corneal edema, inflammation, and thickness using slit-lamp examination, photography, and pachymetry. Rabbits were euthanized and enucleated for further evaluation. Results: H-1152 exhibited significant stimulatory effect on CEC migration and proliferation in vitro compared with both untreated and Y-27632-treated cells. Furthermore, topical administration of H-1152 led to marked reduction in corneal edema and formation of multinucleate CECs in vivo suggestive of proliferation associated with healing. Conclusions: H-1152 exhibited a better stimulatory effect on CEC migration and proliferation in vitro than Y-27632. Our findings suggest that topical administration of H-1152 promotes healing of injured corneal endothelium in vivo. These results demonstrate the efficacy of ROCK inhibitors as a potential topical therapy for patients with corneal endothelial disease.

Growth Differentiation Factor-15-Induced Contractile Activity and Extracellular Matrix Production in Human Trabecular Meshwork Cells.

Purpose: To determine the role and regulation of growth differentiation factor-15 (GDF-15), a TGF-ß-related cytokine in human trabecular meshwork (TM) cells in the context of aqueous humor (AH) outflow and IOP. Methods: Regulation of expression by external cues, and the distribution and secretion of GDF-15 by human TM primary cell cultures, and the effects of recombinant (r) GDF-15 on TM cell contractile characteristics, actin cytoskeleton, cell adhesion, extracellular matrix (ECM), α-smooth muscle actin (αSMA), SMAD signaling, and gene expression were determined by immunoblot, immunofluorescence, mass spectrometry, cDNA microarray, and real-time quantitative PCR (RT-qPCR) analyses. Results: Growth differentiation factor-15, a common constituent of ECM derived from the human TM cells, was confirmed to be distributed throughout the conventional aqueous humor outflow pathway of the human eye. Growth differentiation factor-15 protein levels were significantly increased in human TM cells in response to TGF-ß2, dexamethasone, endothelin-1, lysophosphatidic acid, TNF-α, IL-1ß treatment, and by cyclic mechanical stretch. Stimulation of human TM cells with rGDF-15 caused a significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, SMAD signaling, gene expression, and the levels of αSMA and ECM proteins. Conclusions: The results of this study, including a robust induction of GDF-15 expression by several external factors known to elevate IOP, and rGDF-15-induced increase in contractility, cell adhesion, and the levels of ECM proteins and αSMA in TM cells, collectively suggest a potential role for GDF-15 in homeostasis and dysregulation of AH outflow and IOP in normal and glaucomatous eyes, respectively.

Ankyrin-B in lens architecture and biomechanics: Just not tethering but more.

The ankyrins are a family of well-characterized metazoan adaptor proteins that play a key role in linking various membrane-spanning proteins to the underlying spectrin-actin cytoskeleton; a mechanistic understanding of their role in tissue architecture and mechanics, however, remains elusive. Here we comment on a recent study demonstrating a key role for ankyrin-B in maintaining the hexagonal shape and radial alignment of ocular lens fiber cells by regulating the membrane organization of periaxin, dystrophins/dystroglycan, NrCAM and spectrin-actin network of proteins, and revealing that ankyrin-B deficiency impairs fiber cell shape and mechanical properties of the ocular lens. These observations indicate that ankyrin-B plays an important role in maintaining tissue cytoarchitecture, cell shape and biomechanical properties via engaging in key protein: protein interactions required for membrane anchoring and organization of the spectrin-actin skeleton, scaffolding proteins and cell adhesive proteins.

Ankyrin-B directs membrane tethering of periaxin and is required for maintenance of lens fiber cell hexagonal shape and mechanics.

Periaxin (Prx), a PDZ domain protein expressed preferentially in myelinating Schwann cells and lens fibers, plays a key role in membrane scaffolding and cytoarchitecture. Little is known, however, about how Prx is anchored to the plasma membrane. Here we report that ankyrin-B (AnkB), a well-characterized adaptor protein involved in linking the spectrin-actin cytoskeleton to integral membrane proteins, is required for membrane association of Prx in lens fibers and colocalizes with Prx in hexagonal fiber cells. Under AnkB haploinsufficiency, Prx accumulates in the soluble fraction with a concomitant loss from the membrane-enriched fraction of mouse lenses. Moreover, AnkB haploinsufficiency induced age-dependent disruptions in fiber cell hexagonal geometry and radial alignment and decreased compressive stiffness in mouse lenses parallel to the changes observed in Prx null mouse lens. Both AnkB- and Prx-deficient mice exhibit disruptions in membrane organization of the spectrin-actin network and the dystrophin-glycoprotein complex in lens fiber cells. Taken together, these observations reveal that AnkB is required for Prx membrane anchoring and for maintenance of lens fiber cell hexagonal geometry, membrane skeleton organization, and biomechanics.

Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis.

Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and ß1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis.

Regulation of plasticity and fibrogenic activity of trabecular meshwork cells by Rho GTPase signaling.

Glaucoma, a prevalent blinding disease is commonly associated with increased intraocular pressure due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although increased TM tissue contraction and stiffness in association with accumulation of extracellular matrix (ECM) are believed to be partly responsible for increased resistance to AH outflow, the extracellular cues and intracellular mechanisms regulating TM cell contraction and ECM production are not well defined. This study tested the hypothesis that sustained activation of Rho GTPase signaling induced by lysophosphatidic acid (LPA), TGF-ß, and connective tissue growth factor (CTGF) influences TM cell plasticity and fibrogenic activity which may eventually impact resistance to AH outflow. Various experiments performed using human TM cells revealed that constitutively active RhoA (RhoAV14), TGF-ß2, LPA, and CTGF significantly increase the levels and expression of Fibroblast Specific Protein-1 (FSP-1), α-smooth muscle actin (αSMA), collagen-1A1 and secretory total collagen, as determined by q-RT-PCR, immunofluorescence, immunoblot, flow cytometry and the Sircol assay. Significantly, these changes appear to be mediated by Serum Response Factor (SRF), myocardin-related transcription factor (MRTF-A), Slug, and Twist-1, which are transcriptional regulators known to control cell plasticity, myofibroblast generation/activation and fibrogenic activity. Additionally, the Rho kinase inhibitor-Y27632 and anti-fibrotic agent-pirfenidone were both found to suppress the TGF-ß2-induced expression of αSMA, FSP-1, and collagen-1A1. Taken together, these observations demonstrate the significance of RhoA/Rho kinase signaling in regulation of TM cell plasticity, fibrogenic activity, and myofibroblast activation, events with potential implications for the pathobiology of elevated intraocular pressure in glaucoma patients.