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.

Elevated intraocular pressure induces Rho GTPase mediated contractile signaling in the trabecular meshwork.

Rho GTPase regulated contractile signaling in the trabecular meshwork (TM) has been shown to modulate aqueous humor (AH) outflow and intraocular pressure (IOP). To explore whether elevated IOP, a major risk factor for primary open angle glaucoma (POAG) influences Rho GTPase signaling in the TM, we recorded AH outflow in enucleated contralateral porcine eyes perfused for 4-5 h at either 15 mm or 50 mm Hg pressure. After perfusion, TM tissue extracted from perfused eyes was evaluated for the activation status of Rho GTPase, myosin light chain (MLC), myosin phosphatase target substrate 1 (MYPT1), myristoylated alanine-rich C-kinase substrate (MARCKS) and paxillin. Eyes perfused at 50 mm Hg exhibited a significant decrease in AH outflow facility compared with those perfused at 15 mm Hg. Additionally, TM tissue from eyes perfused at 50 mm Hg revealed significantly increased levels of activated RhoA and phosphorylated MLC, MYPT1, MARCKS and paxillin compared to TM tissue derived from eyes perfused at 15 mm Hg. Taken together, these observations indicate that elevated IOP-induced activation of Rho GTPase-dependent contractile signaling in the TM is associated with increased resistance to AH outflow through the trabecular pathway, and demonstrate the sensitivity of Rho GTPase signaling to mechanical force in the AH outflow pathway.

Characterization of cytoskeleton-enriched protein fraction of the trabecular meshwork and ciliary muscle cells.

PURPOSE: To understand the molecular basis for the known distinct contractile characteristics of trabecular meshwork (TM) and ciliary muscle (CM) cells, the cytoskeleton-enriched protein fractions of the TM and CM cells were isolated and characterized. METHODS: The nonionic surfactant insoluble fraction enriched for cytoskeletal proteins was isolated from human and porcine TM tissue and cells and from CM cells and was characterized by SDS-PAGE, mass spectrometry, and immunoblotting techniques. RESULTS: The cytoskeleton-enriched protein fraction derived from both human and porcine TM cells contained Plectin 1, Filamin A, non-muscle myosin IIA, clathrin, α-actinin, vimentin, actin, caldesmon, myosin IC, and annexin A2 as major proteins and was noted to exhibit compositional similarity with the cytoskeletal protein fraction isolated from TM tissue. Importantly, the cytoskeletal protein composition of the TM cells was also found to be similar to that noted for CM and vascular endothelial cells. Although the activity of myosin II, a crucial regulator of cellular contraction and a major component of the cytoskeletal protein fraction in TM and CM cells, was regulated predominantly by Rho kinase in both cell types, myosin light chain kinase (MLCK) also appeared to control myosin II activity in CM cells. CONCLUSIONS: These data reveal that the activity of non-muscle myosin II, a critical molecule of cellular contraction, was found to be regulated differentially in TM and CM cells by the Rho kinase and the MLCK pathways despite their compositional similarity in cytoskeletal protein profile.

Abundant expression of ponsin, a focal adhesion protein, in lens and downregulation of its expression by impaired cytoskeletal signaling.

PURPOSE: This study was undertaken to improve understanding of the defective lens developmental changes induced by the transgenic overexpression of the Rho GDP dissociation inhibitor RhoGDIalpha. The study was focused on a single differentially expressed gene encoding ponsin, a cell adhesion interacting signaling adaptor protein. METHODS: Total RNA extracted from the P7 lenses of Rho GDIalpha transgenic mice was subjected to cDNA microarray analysis. Ponsin distribution in the mouse lenses was determined by immunofluorescence and immunoblot analyses. Interactions among ponsin, actin, and Rho GTPase signaling pathways were explored in lens epithelial cells. RESULTS: The RhoGDIalpha transgenic mouse lenses revealed a marked downregulation of expression of multiple splice variants of ponsin. Expression of one of the ponsins (U58883) was found to be abundant in normal mouse lenses. Although ponsin was localized predominantly to the focal adhesions in lens epithelial cells, it was distributed to both the epithelium and fibers, with some isoforms being enriched primarily in the Triton X-100-insoluble fraction in lens tissue. Further, whereas constitutively active RhoA induced ponsin clustering at the leading edges, inhibition of Rho kinase and latrunculin treatment were noted to lead to decreases in ponsin protein levels in lens epithelial cells. CONCLUSIONS: Abundant expression of ponsin, a focal adhesion protein in the lens tissue indicates a potential role for this protein in lens fiber cell migration and adhesion. Ponsin expression appears to be closely dependent on Rho GTPase-regulated integrity of actin cytoskeletal organization.

Effects of pharmacologic inhibition of protein geranylgeranyltransferase type I on aqueous humor outflow through the trabecular meshwork.

PURPOSE: To determine the effects of inhibition of protein geranylgeranyltransferase type I (GGTase-I), which isoprenylates so-called CaaX proteins, including the GTP-binding proteins such as Rho GTPases and the betagamma subunits of heterotrimeric G-proteins, on aqueous humor outflow and trabecular meshwork cytoskeletal integrity. METHODS: A selective small molecular inhibitor of GGTase-I, GGTI-DU40, was tested in this study to investigate its effects on actin cytoskeletal integrity, cell adhesions, cell-cell junctions, myosin II phosphosphorylation, and membrane localization of GTP-binding proteins in trabecular meshwork (TM) cells, using immunofluorescence detection and immunoblotting analysis. The effects of GGTI-DU40 on aqueous humor outflow were determined using organ-cultured, perfused anterior segments of porcine eyes. RESULTS: In the TM cell lysates, GGTI-DU40 was confirmed to inhibit GGTase-I activity in a dose-dependent manner. TM cells treated with GGTI-DU40 displayed dose-dependent changes in cell morphology and reversible decreases in actin stress fibers, focal adhesions, and adherens junctions. Myosin light chain phosphorylation was decreased significantly, and membrane localization of isoprenylated small GTPases and Gbetagamma was impaired in drug-treated TM cells. Aqueous outflow facility was increased significantly in eyes perfused with GGTI-DU40. CONCLUSIONS: These data demonstrate that inhibition of geranylgeranyl isoprenylation of CaaX proteins in the aqueous outflow pathway increases aqueous humor outflow, possibly through altered cell adhesive interactions and actin cytoskeletal organization in cells of the outflow pathway. This study indicates that the GGTase-I enzyme is a promising molecular target for lowering increased ocular pressure in glaucoma patients.

Characterization of lens fiber cell triton insoluble fraction reveals ERM (ezrin, radixin, moesin) proteins as major cytoskeletal-associated proteins.

To understand lens fiber cell elongation- and differentiation-associated cytoskeletal 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 beta-actin as major cytoskeletal proteins in the lens fibers. Importantly, ezrin, radixin, and moesin (ERM), heat-shock cognate protein 70, and beta/gamma-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 GTPase-targeted transgenic mouse lenses. These data identify the ERM proteins, which cross-link 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.

The pulling, pushing and fusing of lens fibers: a role for Rho GTPases.

Lens development and differentiation are intricate and complex processes characterized by distinct molecular and morphological changes. The growth of a transparent lens involves proliferation of the epithelial cells and their subsequent differentiation into secondary fiber cells. Prior to differentiation, epithelial cells at the lens equator exit from the cell cycle and elongate into long, ribbon-like cells. Fiber cell elongation takes place bidirectionally as fiber tips migrate both anteriorly and posteriorly along the apical surface of the epithelium and inner surface of the capsule, respectively. The differentiating fiber cells move inward from the periphery to the center of the lens on a continuous basis as the lens grows throughout life. Finally, when fiber cells reach the center or suture line, their basal and apical tips detach from the epithelium and capsule, respectively, and interlock with cells from the opposite direction of the lens and form the suture line. Further, symmetric packing of fiber cells and degradation of most of the cellular organelle during fiber cell terminal differentiation are crucial for lens transparency. These sequential events are presumed to depend on cytoskeletal dynamics and cell adhesive interactions; however, our knowledge of regulation of lens fiber cell cytosketal reorganization, cell adhesive interactions and mechanotransduction, and their role in lens morphogenesis and function is limited at present. Recent biochemical and molecular studies have targeted cytoskeletal signaling proteins, including Rho GTPases, Abl kinase interacting proteins, cell adhesion molecules, myosin II, Src kinase and phosphoinositide 3-kinase in the developing chicken and mouse lens and characterized components of the fiber cell basal membrane complex. These studies have begun to unravel the vital role of cytoskeletal proteins and their regulatory pathways in control of lens morphogenesis, fiber cell elongation, migration, differentiation, survival and mechanical properties.

Influence of actin cytoskeletal integrity on matrix metalloproteinase-2 activation in cultured human trabecular meshwork cells.

PURPOSE: The goal of this study was to investigate the possible link between actin cytoskeletal integrity and the activation of matrix metalloproteinases (MMPs) in trabecular meshwork (TM) cells. METHODS: Primary human TM (HTM) cells treated with different actin cytoskeleton-interfering agents, including cytochalasin D, latrunculin A, ethacrynic acid (ECA), a Rho kinase inhibitor (Y-27632), and H-7 (serine/threonine kinase inhibitor), were examined for changes in actin cytoskeletal organization by phalloidin staining, MMP-2 activation by gelatin zymography, expression of MT1-MMP by quantitative real-time PCR analysis, levels of tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2), and activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated protein kinase (ERK) by immunoblotting. RESULTS: Treatment of HTM cells with cytochalasin D and latrunculin A led to significant activation of MMP-2, p38 MAPK, and ERK1/2, which appeared to correlate with changes in cell morphology and actin depolymerization. Additionally, treatment with these cytoskeleton-disrupting agents elicited increased expression of MT1-MMP in HTM cells, concomitant with a decrease in the levels of secreted TIMP-1 and TIMP-2. In contrast, treatment with ECA, Y-27632, or H-7 triggered changes in cell shape and reduced actin stress fibers in HTM cells but did not exert significant effects on MMP-2 activation or MT1-MMP expression. CONCLUSIONS: These studies indicate that cytochalasin D- and latrunculin A-induced alteration of actin cytoskeletal integrity in HTM cells is associated with MMP-2 activation, most likely through the upregulation of its activator, MT1-MMP. These data provide a mechanistic connection between actin cytoskeletal organization and MMP-2 activation in TM cells and offer new insights into extracellular matrix remodeling in the aqueous outflow pathway.

The role of the lens actin cytoskeleton in fiber cell elongation and differentiation.

The vertebrate ocular lens is a fascinating and unique transparent tissue that grows continuously throughout life. During the process of differentiation into fiber cells, lens epithelial cells undergo dramatic morphological changes, membrane remodeling, polarization, transcriptional activation and elimination of cellular organelles including nuclei, concomitant with migration towards the lens interior. Most of these events are presumed to be influenced in large part, by dynamic reorganization of the cellular actin cytoskeleton and by intercellular and cell: extracellular matrix interactions. In light of recent and unprecedented advancement in our understanding of the mechanistic bases underlying regulation of actin cytoskeletal dynamics and the role of the actin cytoskeleton in cell function, this review attempts to summarize current knowledge regarding the role of the cellular actin cytoskeleton, in lens fiber cell elongation and differentiation, and regulation of actin cytoskeletal organization in the lens.

Regulation of connective tissue growth factor expression in the aqueous humor outflow pathway.

PURPOSE: Connective Tissue Growth Factor (CTGF) is an inducible secretory protein known to regulate proliferation and extracellular matrix production in various cell types. We hypothesize that CTGF plays a critical role in the physiological regulation of aqueous humor outflow through the trabecular meshwork (TM) by influencing extracellular matrix synthesis and organization. METHODS: To determine the expression of CTGF in tissues of the aqueous outflow pathway, cells obtained from human TM and Schlemm's Canal (SC) were analyzed by PCR and western blot analysis. To understand the regulation of CTGF expression in TM cells, TM cells were either treated with various physiologic factors or subjected to cyclical stretch prior to analysis of CTGF expression by RT-PCR and western blot analysis. To study the effect of increased intraocular pressure on CTGF production, we perfused porcine eyes at high pressure (50 mm Hg) for 5 h, followed by analysis of CTGF expression by RT-PCR and western blotting. RESULTS: Treatment of human TM cells treated with either serum or transforming growth factor-beta 1 led to a robust stimulation, compared to thrombin, lysophosphatidic acid (LPA), and dexamethasone, which elicited a relatively moderate induction of CTGF expression. Both high pressure perfusion and mechanical stretch were associated with increases in the levels of CTGF at the protein and transcript levels. CONCLUSIONS: This study demonstrates that CTGF expression in TM cells is modulated by several physiological agonists and by increased ocular pressure and mechanical stretch. These results suggest that the regulation of CTGF expression within tissues of the outflow pathway may play a role in the homeostasis of intraocular pressure, possibly by modulation of ECM production in these tissues.

Blebbistatin, a novel inhibitor of myosin II ATPase activity, increases aqueous humor outflow facility in perfused enucleated porcine eyes.

PURPOSE: To investigate the specific role of myosin II, a critical biochemical determinant of cellular contraction, in modulation of aqueous humor outflow facility through the trabecular meshwork (TM) pathway. METHODS: Expression of the nonmuscle myosin II heavy chains (IIA, IIB, and IIC) in human TM and ciliary body (CB) cells was determined by RT-PCR analyses. The effects of inhibition of myosin II on cell morphology, actomyosin organization, and cell adhesions were evaluated in porcine TM and CB cells treated with blebbistatin, a cell-permeable, specific inhibitor of myosin II adenosine triphosphatase (ATPase) activity. Changes in aqueous humor outflow facility were determined in enucleated porcine eyes by using a constant-pressure Grant perfusion model system. Ultrastructural integrity of the outflow pathway in drug-perfused eyes was analyzed by transmission electron microscopy. RESULTS: Expression of nonmuscle myosin IIA and IIB was confirmed in both human TM and CB cells. Confluent cultures of primary porcine TM and CB cells treated with blebbistatin in the presence of serum revealed dose (10-200 microM)-dependent changes in cell morphology, decreases in actin stress fiber content and in focal adhesions and adherens junctions. These changes were found to be reversible within 24 hours of drug withdrawal from the cell culture media. Blebbistatin did not affect the status of myosin light chain phosphorylation in TM cells. Perfusion of enucleated porcine eyes for 5 hours with 100 and 200 microM blebbistatin produced a significant increase (P < 0.01, n = 7) in aqueous outflow facility (53% and 64%, respectively) from the baseline facility, compared with a 21% facility increase in sham control specimens. The integrity of the inner wall of aqueous plexi in drug-perfused porcine eyes was found to be intact, and TM cell morphology appeared to be similar to that noted in sham-treated eyes. CONCLUSIONS: These data demonstrate that selective inhibition of myosin II in the aqueous humor outflow pathway leads to increased aqueous outflow facility, suggesting a critical role for myosin II in the regulation of aqueous humor outflow facility. This study also suggests myosin II as a potential therapeutic target for lowering intraocular pressure in patients with glaucoma.

Effects of cholesterol-lowering statins on the aqueous humor outflow pathway.

PURPOSE: To investigate the effects of cholesterol-lowering statin drugs on trabecular meshwork cellular properties and aqueous humor outflow. METHODS: Primary cell cultures of porcine trabecular meshwork (PTM) and ciliary body (PCB) were treated with either lovastatin or compactin, to determine the effects of statins on cell shape, actin cytoskeletal organization, and cell-extracellular matrix interactions (focal adhesions) by immunofluorescence staining. Changes in myosin light-chain (MLC) phosphorylation were evaluated by Western blot analysis. Changes in Rho GTPase content of membrane fractions from lovastatin-treated PTM cells were assessed by Western blot analysis. A constant-flow, organ-culture perfusion system was used to measure the effects of statins on aqueous humor outflow facility in the anterior segments of porcine eyes. RESULTS: PTM and PCB cells treated with lovastatin or compactin exhibited dramatic changes in cell shape and cytoskeletal organization within 24 hours, consisting of cell rounding, actin depolymerization, and decreased focal adhesions. These effects were found to be reversible on supplementation with geranylgeranyl pyrophosphate. Both lovastatin and compactin decreased MLC phosphorylation in PTM and PCB cells. PTM cells treated with lovastatin exhibited marked decreases in membrane-bound Rho GTPase. In addition, perfusion of organ-cultured porcine eye anterior segments with 100 microM lovastatin for 96 hours caused a significant increase in aqueous humor outflow facility (110%) compared with control eyes, in a reversible manner. CONCLUSIONS: This study demonstrates that the statin drugs lovastatin and compactin induce changes in cell shape and actin cytoskeletal organization and decrease MLC phosphorylation in PTM and PCB cells, all of which are events that are likely to lead to cellular and tissue relaxation. In addition, these effects of the statins appear to be mediated by inhibition of isoprenylation of the small GTP-binding proteins such as Rho GTPase. An important finding is that statins exert an ocular hypotensive response in an organ-culture perfusion model, indicating the potential for this class of drugs in glaucoma therapy.

Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humour outflow facility.

Cellular contraction and relaxation and integrity of the actin cytoskeleton in trabecular meshwork (TM) tissue have been thought to influence aqueous humour outflow. However, the cellular pathways that regulate these events in TM cells are not well understood. In this study, we investigated physiological agonist-mediated regulation of myosin light chain (MLC) phosphorylation in the TM, and correlated such effects with alterations in aqueous outflow facility, since MLC phosphorylation is a critical biochemical determinant of cellular contraction in TM cells. Treatment of serum starved human TM cells with endothelin-1 (0.1 microM), thromboxane A2 mimetic U-46619 (1.0 microM), or angiotensin II (1 microM), all of which are agonists of G-protein coupled receptors, triggered activation of MLC phosphorylation, as determined by urea/glycerol-based Western blot analysis. Agonist-stimulated increase in MLC phosphorylation was associated with activation of Rho GTPase in TM cells, as determined in pull-down assays. In contrast, treatment of human TM cells with a novel Rho-kinase inhibitor H-1152 (0.1-2 microM), in the presence of serum reduced basal MLC phosphorylation. H-1152 also increased aqueous outflow facility significantly in a dose-dependent fashion, in perfusion studies with cadaver porcine eyes. This effect of H-1152 on outflow facility was associated with decreased MLC phosphorylation in TM tissue of drug-perfused eyes. Collectively, this study identifies potential physiological regulators of MLC phosphorylation in human TM cells and demonstrates the significance of Rho/Rho-kinase pathway-mediated MLC phosphorylation in modulation of aqueous outflow facility through TM.

Role of lysophospholipid growth factors in the modulation of aqueous humor outflow facility.

PURPOSE: To investigate the role of lysophospholipid growth factors in the regulation of aqueous humor outflow in the trabecular meshwork (TM). METHODS: The expression profile of the endothelial differentiation gene (Edg) family of G-protein coupled receptors was determined by RT-PCR of human TM (HTM) cell-derived total RNA and by PCR amplification of HTM cell-derived and tissue-derived cDNA libraries. The effects of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) on actin cytoskeleton and focal adhesions and on myosin light-chain (MLC) phosphorylation in HTM cells were evaluated by immunofluorescence microscopy and Western blot analysis, respectively. Activation of Rho GTPase in HTM cells was quantified by "pull-down" assays. Mobilization of intracellular calcium in HTM cells was determined using spectrofluorometric digital-imaging microscopy. The effects of LPA and S1P on aqueous humor outflow facility were evaluated by perfusion of enucleated porcine eyes. RESULTS: Each of the receptor isoforms Edg1, -2, -3, and -4 was readily detectable in three of four HTM cell-derived libraries, whereas Edg2 was detectable in the HTM tissue library. LPA (20 microM) and S1P (1 microM) stimulated actin stress fiber and focal adhesion formation, increased MLC phosphorylation, and induced marked activation of Rho GTPase in HTM cells. Both LPA (20 microM) and S1P (10 microM) also stimulated increases in intracellular calcium concentration in HTM cells. LPA- and S1P-induced effects on MLC phosphorylation in HTM cells were markedly inhibited by pretreatment with the Rho kinase-specific inhibitor Y-27632 (5 microM). Perfusion of LPA (50 microM) and S1P (5 microM) in enucleated porcine eyes produced a significant decrease in aqueous humor outflow facility from baseline of 37% (n = 6) and 31% (n = 5), respectively. CONCLUSIONS: These studies demonstrate that LPA and S1P, the physiological agonists of Edg receptors, decrease outflow facility in perfused porcine eyes in association with increased MLC phosphorylation and Rho guanosine triphosphatase (GTPase) activation. These data provide evidence for a novel mechanism for negative regulation of outflow facility, which may contribute to overall physiological homeostasis of aqueous humor outflow facility.