Ponugoti Vasantha Rao

Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells

Elevated intraocular pressure arising from impaired aqueous humor drainage through the trabecular pathway is a major risk factor for glaucoma. To understand the molecular basis for Rho GTPase-mediated resistance to aqueous humor drainage, we investigated the possible interrelationship between actomyosin contractile properties and extracellular matrix (ECM) synthesis in human trabecular meshwork (TM) cells expressing a constitutively active form of RhoA (RhoAV14). TM cells expressing RhoAV14 exhibited significant increases in fibronectin, tenascin C, laminin, alpha-smooth muscle actin (alpha-SMA) levels, and matrix assembly in association with increased actin stress fibers and myosin light-chain phosphorylation. RhoAV14-induced changes in ECM synthesis and actin cytoskeletal reorganization were mimicked by lysophosphatidic acid and TGF-beta(2), known to increase resistance to aqueous humor outflow and activate Rho/Rho kinase signaling. RhoAV14, lysophosphatidic acid, and TGF-beta(2) stimulated significant increases in Erk1/2 phosphorylation, paralleled by profound increases in fibronectin, serum response factor (SRF), and alpha-SMA expression. Treatment of RhoA-activated TM cells with inhibitors of Rho kinase or Erk, on the other hand, decreased fibronectin and alpha-SMA levels. Although suppression of SRF expression (both endogenous and RhoA, TGF-beta(2)-stimulated) via the use of short hairpin RNA decreased alpha-SMA levels, fibronectin was unaffected. Conversely, fibronectin induced alpha-SMA expression in an SRF-dependent manner. Collectively, data on RhoA-induced changes in actomyosin contractile activity, ECM synthesis/assembly, and Erk activation, along with fibronectin-induced alpha-SMA expression in TM cells, reveal a potential molecular interplay between actomyosin cytoskeletal tension and ECM synthesis/assembly. This interaction could be significant for the homeostasis of aqueous humor drainage through the pressure-sensitive trabecular pathway.

Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells.

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‐β, 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‐β2, 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‐β2‐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. J. Cell. Physiol. 229: 927–942, 2014.

Periaxin is required for hexagonal geometry and membrane organization of mature lens fibers

Transparency of the ocular lens depends on symmetric packing and membrane organization of highly elongated hexagonal fiber cells. These cells possess an extensive, well-ordered cortical cytoskeleton to maintain cell shape and to anchor membrane components. Periaxin (Prx), a PDZ domain protein involved in myelin sheath stabilization, is also a component of adhaerens plaques in lens fiber cells. Here we show that Prx is expressed in lens fibers and exhibits maturation dependent redistribution, clustering discretely at the tricellular junctions in mature fiber cells. Prx exists in a macromolecular complex with proteins involved in membrane organization including ankyrin-B, spectrin, NrCAM, filensin, ezrin and desmoyokin. Importantly, Prx knockout mouse lenses were found to be softer and more easily deformed than normal lenses, revealing disruptions in fiber cell hexagonal packing, membrane skeleton and membrane stability. These observations suggest a key role for Prx in maturation, packing, and membrane organization of lens fiber cells. Hence, there may be functional parallels between the roles of Prx in membrane stabilization of the myelin sheath and the lens fiber cell.

Enzyme/crystallins and extremely high pyridine nucleotide levels in the eye lens.

Taxon‐specific crystalling are proteins present in high abundance in the lens of phylogenetically restricted groups of animals. Recently it has been found that these proteins are actually enzymes which the lens has apparently adopted to serve as structural proteins. Most of these proteins have been shown to be identical to, or related to, oxidoreductases. In guinea pig lens, which contains zeta‐crystallin, a protein with an NADPH‐dependent oxidoreductase activity, the levels of both NADPH and NADP+ are extremely high and correlate with the concentration of zeta‐crystallin. We report here nucleotide assays on lenses from vertebrates containing other enzyme/crystallins. In each case where the enzyme/crystallin is a pyridine nucleotide‐binding protein the level of that particular nucleotide is extremely high in the lens. The presence of an enzyme/crystallin does not affect the lenticular concentrations of those nucleotides which are not specifically bound. The possibility that nucleotide binding may be a factor in the selection of some enzymes to serve as enzyme/crystallins is considered.—Zigler, J. S., Jr.; Rao, P. V. Enzyme/crystallins and extremely high pyridine nucleotide levels in the eye lens. FASEB J. 5: 223–225; 1991.

Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival

Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell-cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover.

Autotaxin-Lysophosphatidic Acid Axis Is a Novel Molecular Target for Lowering Intraocular Pressure

Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ∼1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity in vitro (by >90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.

Rho GDP Dissociation Inhibitor-Mediated Disruption of Rho GTPase Activity Impairs Lens Fiber Cell Migration, Elongation and Survival

To explore the role of the Rho GTPases in lens morphogenesis, we overexpressed bovine Rho GDP dissociation inhibitor (Rho GDI alpha), which serves as a negative regulator of Rho, Rac and Cdc42 GTPase activity, in a lens-specific manner in transgenic mice. This was achieved using a chimeric promoter of delta-crystallin enhancer and alpha A-crystallin, which is active at embryonic day 12. Several individual transgenic (Tg) lines were obtained, and exhibited ocular specific phenotype comprised of microphthalmic eyes with lens opacity. The overexpression of bovine Rho GDI alpha disrupted membrane translocation of Rho, Rac and Cdc42 GTPases in Tg lenses. Transgenic lenses also revealed abnormalities in the migration pattern, elongation and organization of lens fibers. These changes appeared to be associated with impaired organization of the actin cytoskeleton and cell-cell adhesions. At E14.5, the size of the Rho GDI alpha Tg lenses was larger compared to wild type (WT) and the central lens epithelium and differentiating fibers exhibited an abnormal increase of bromo-deoxy-uridine incorporation. Postnatal Tg eyes, however, were much smaller in size compared to WT eyes, revealing increased apoptosis in the disrupted lens fibers. Taken together, these data demonstrate a critical role for Rho GTPase-dependent signaling pathways in processes underlying morphogenesis, fiber cell migration, elongation and survival in the developing lens.

Connective tissue growth factor-mediated upregulation of neuromedin U expression in trabecular meshwork cells and its role in homeostasis of aqueous humor outflow.

PURPOSE Connective tissue growth factor (CTGF) is a matricellular protein presumed to be involved in the pathobiology of various fibrotic diseases, including glaucoma. We investigated the effects of Rho GTPase-dependent actin cytoskeletal integrity on CTGF expression and CTGF-induced changes in gene expression profile in human trabecular meshwork (HTM) cells. METHODS CTGF levels were quantified by immunoblotting and ELISA. CTGF-induced changes in gene expression, actin cytoskeleton, myosin light chain (MLC) phosphorylation, and extracellular matrix (ECM) proteins were evaluated in trabecular meshwork (TM) cells by cDNA microarray, q-PCR, fluorescence microscopy, and immunoblot analyses. The effects of neuromedin U (NMU) on aqueous humor (AH) outflow were determined in enucleated porcine eyes. RESULTS Expression of a constitutively active form of RhoA (RhoAV14), activation of Rho GTPase by bacterial toxin, or inhibition of Rho kinase by Y-27632 in HTM cells led to significant but contrasting changes in CTGF protein levels that were detectable in cell lysates and cell culture medium. Stimulation of HTM cells with CTGF for 24 hours induced actin stress fiber formation, and increased MLC phosphorylation, fibronectin, and laminin levels, and NMU expression. NMU independently induced actin stress fibers and MLC phosphorylation in TM cells, and decreased AH outflow facility in perfused porcine eyes. CONCLUSIONS These data revealed that CTGF influences ECM synthesis, actin cytoskeletal dynamics, and contractile properties in TM cells, and that the expression of CTGF is regulated closely by Rho GTPase. Moreover, NMU, whose expression is induced in response to CTGF, partially mimics the effects of CTGF on actomyosin organization in TM cells, and decreases AH outflow facility, revealing a potentially important role for this neuropeptide in the homeostasis of AH drainage.

Elevated levels of RhoA in the optic nerve head of human eyes with glaucoma.

Purpose:To determine changes in the expression profile of RhoA and Rho kinase (ROCK-1 and ROCK-2) in the aqueous humor outflow pathway and optic nerve head (ONH) of human eyes with or without glaucoma to explore their potential involvement in glaucoma pathophysiology. Methods:Age-matched paraffin-embedded postmortem eyes from patients with or without glaucoma were stained immunohistochemically using polyclonal antibodies raised against RhoA, ROCK-1, and ROCK-2. The intensity of the immunostaining in the aqueous humor outflow pathway and the ONH was graded by 4 individuals who were masked concerning whether the eyes were from normal individuals or those with glaucoma. Results:Both normal eyes and those with glaucoma showed a positive staining for RhoA, ROCK-1, and ROCK-2 in the trabecular meshwork, ciliary muscle, and ONH. There was a significant increase in the RhoA protein levels in the glaucomatous ONH compared with the age-matched controls. Conclusions:Elevated levels of RhoA in the ONH of glaucomatous eyes suggest possible involvement of RhoA in the pathophysiology of glaucoma.