Myocilin binding to Hep II domain of fibronectin inhibits cell spreading and incorporation of paxillin into focal adhesions

Myocilin, a novel matricellular protein found in the human eye, can modify signaling events mediated by the Heparin II domain of fibronectin. Using myocilin produced in sf9 insect cells, myocilin inhibited spreading of cycloheximide-treated human skin fibroblasts plated on substrates co-coated with myocilin and either fibronectin or its Heparin II domain. Cell spreading could be rescued by adding back either substrate adsorbed or soluble Heparin II domains. Myocilin did not inhibit cell attachment to fibronectin even in the presence of a 2400 M excess of myocilin. Myocilin impaired focal adhesion formation and specifically blocked the incorporation of paxillin, but not vinculin, into focal adhesions. The Heparin II domain mediated the incorporation of paxillin into focal adhesions, since paxillin was not assembled into focal adhesions unless the Heparin II domain was present. The effect of myocilin on focal adhesions could be overcome by treating cells with either phorbol 12-myristate (PMA) or oleoyl-L-alpha-lysophosphatidic acid (LPA). Myocilin bound to the fibroblast cell surface, but its binding could not be competed with excess fibronectin, suggesting that myocilin does not compete for cell surface binding sites of fibronectin. Myocilin therefore appears to specifically block functions mediated by the Heparin II domain possibly through direct interactions with it.     

Amyloid fibril formation by the glaucoma-associated olfactomedin domain of myocilin

Myocilin is a protein found in the extracellular matrix of trabecular meshwork tissue, the anatomical region of the eye involved in regulating intraocular pressure. Wild-type (WT) myocilin has been associated with steroid-induced glaucoma, and variants of myocilin have been linked to early-onset inherited glaucoma. Elevated levels and aggregation of myocilin hasten increased intraocular pressure and glaucoma-characteristic vision loss due to irreversible damage to the optic nerve. In spite of reports on the intracellular accumulation of mutant and WT myocilin in vitro, cell culture, and model organisms, these aggregates have not been structurally characterized. In this work, we provide biophysical evidence for the hallmarks of amyloid fibrils in aggregated forms of WT and mutant myocilin localized to the C-terminal olfactomedin (OLF) domain. These fibrils are grown under a variety of conditions in a nucleation-dependent and self-propagating manner. Protofibrillar oligomers and mature amyloid fibrils are observed in vitro. Full-length mutant myocilin expressed in mammalian cells forms intracellular amyloid-containing aggregates as well. Taken together, this work provides new insights into and raises new questions about the molecular properties of the highly conserved OLF domain, and suggests a novel protein-based hypothesis for glaucoma pathogenesis for further testing in a clinical setting.     

Extracellular myocilin affects activity of human trabecular meshwork cells

The trabecular meshwork (TM), a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of this tissue is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. The protein product has been localized to both intra and extracellular sites, but its function still remains unclear. This study was to determine whether extracellular myocilin presented in the matrix affects adhesion, morphology, and migratory and phagocytic activities of human TM cells in culture. Cell adhesion assays indicated that TM cells, while adhering readily on fibronectin, failed to attach on recombinant myocilin purified from bacterial cultures. Adhesion on fibronectin was also compromised by myocilin in a dose dependent manner. Myocilin in addition triggered TM cells to assume a stellate appearance with broad cell bodies and microspikes. Loss of actin stress fibers and focal adhesions was observed. TM cell migration on fibronectin/myocilin to scratched wounds was reduced compared to fibronectin controls. Myocilin, however, had little impact on phagocytic activities of TM cells. Cell attachment on fibronectin and migration of corneal fibroblasts, a control cell type, were not altered by myocilin. These results demonstrate that extracellular myocilin elicits anti-adhesive and counter-migratory effects on TM cells. Myocilin in the matrix of tissues could be exerting a similar influence on TM cells in vivo, impacting the flexibility and resilience required for maintenance of the normal aqueous outflow.     

Cellular immunohistochemical localization of the matricellular protein myocilin in the intervertebral disc

Myocilin is a 55-57-kDa protein that is a member of the olfactomedin protein family. It is expressed in the cornea, sclera and trabecular network of the eye, myelinated peripheral nerves, heart, skeletal muscle, trachea and other tissues. Myocilin binds to a domain of fibronectin, type IV collagen and laminen in the trabecular meshwork of the eye, and its expression is influenced by transforming growth factor beta. Because these extracellular matrix components also are common in the intervertebral disc, the objective of our study was to determine whether the matricellular protein myocilin could be detected in the human or sand rat intervertebral disc using immunohistochemistry and to assess its localization. We investigated 16 specimens of human disc tissue and discs from six sand rats. Three human disc cell cultures grown in three-dimensional culture also were evaluated. Immunocytochemical annulus analysis showed the presence of myocilin within the disc cell cytoplasm in some, but not all, cells. Extracellular matrix in both the human and sand rat disc was negative for myocilin localization. Myocilin is believed to play a role in cell-cell adhesion and/or signaling. Myocilin may have such functions within the disc cell population in a manner similar to tenascin, SPARC and thrombospondin, which are other matricellular proteins recently shown to be present in the disc.     

Cellular immunohistochemical localization of the matricellular protein myocilin in the intervertebral disc

Myocilin is a 55-57-kDa protein that is a member of the olfactomedin protein family. It is expressed in the cornea, sclera and trabecular network of the eye, myelinated peripheral nerves, heart, skeletal muscle, trachea and other tissues. Myocilin binds to a domain of fibronectin, type IV collagen and laminen in the trabecular meshwork of the eye, and its expression is influenced by transforming growth factor beta. Because these extracellular matrix components also are common in the intervertebral disc, the objective of our study was to determine whether the matricellular protein myocilin could be detected in the human or sand rat intervertebral disc using immunohistochemistry and to assess its localization. We investigated 16 specimens of human disc tissue and discs from six sand rats. Three human disc cell cultures grown in three-dimensional culture also were evaluated. Immunocytochemical annulus analysis showed the presence of myocilin within the disc cell cytoplasm in some, but not all, cells. Extracellular matrix in both the human and sand rat disc was negative for myocilin localization. Myocilin is believed to play a role in cell-cell adhesion and/or signaling. Myocilin may have such functions within the disc cell population in a manner similar to tenascin, SPARC and thrombospondin, which are other matricellular proteins recently shown to be present in the disc.     

Cellular immunohistochemical localization of the matricellular protein myocilin in the intervertebral disc.

Myocilin is a 55-57-kDa protein that is a member of the olfactomedin protein family. It is expressed in the cornea, sclera and trabecular network of the eye, myelinated peripheral nerves, heart, skeletal muscle, trachea and other tissues. Myocilin binds to a domain of fibronectin, type IV collagen and laminen in the trabecular meshwork of the eye, and its expression is influenced by transforming growth factor beta. Because these extracellular matrix components also are common in the intervertebral disc, the objective of our study was to determine whether the matricellular protein myocilin could be detected in the human or sand rat intervertebral disc using immunohistochemistry and to assess its localization. We investigated 16 specimens of human disc tissue and discs from six sand rats. Three human disc cell cultures grown in three-dimensional culture also were evaluated. Immunocytochemical annulus analysis showed the presence of myocilin within the disc cell cytoplasm in some, but not all, cells. Extracellular matrix in both the human and sand rat disc was negative for myocilin localization. Myocilin is believed to play a role in cell-cell adhesion and/or signaling. Myocilin may have such functions within the disc cell population in a manner similar to tenascin, SPARC and thrombospondin, which are other matricellular proteins recently shown to be present in the disc.     

Myocilin蛋白在小梁网组织中的作用

Myocilin基因是与原发性开角型青光眼成因有关的基因。其蛋白产物myocilin蛋白是一种分泌型糖蛋白,具有特征性区域：N端亮氨酸拉链区,中央链接区,C端类嗅质蛋白（嗅素）区。眼组织中,小梁网myocilin蛋白表达水平最高且在细胞内外均可检测到。细胞内myocilin蛋白由小梁网细胞以外泌体样囊泡形式释放至胞外,突变时分泌受阻并异常聚集,使细胞致敏诱发凋亡。细胞外myocilin蛋白通过与一种或多种细胞外基质蛋白相互作用影响细胞的形态、粘接、迁移活动,调节细胞外基质的成分和结构,从而影响房水流出系统。     

Rho GTPase and cAMP/protein kinase A signaling mediates myocilin-induced alterations in cultured human trabecular meshwork cells

Myocilin is a gene linked to the most common form of glaucoma, a major blinding disease. The trabecular meshwork (TM), a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The myocilin expression is known to be up-regulated by glucocorticoids in TM cells, and an altered myocilin level may be the culprit in conditions such as corticosteroid glaucoma. Wild type myocilin, when transfected into cultured human TM cells, induced a dramatic loss of actin stress fibers and focal adhesions. Myocilin transfectants displayed a heightened sensitivity to trypsin. Adhesion to fibronectin, collagens, and vitronectin was compromised. The fibronectin deposition and the levels of fibronectin protein and mRNA were also reduced in myocilin transfectants. The fibronectin deposition could be restored by treatment with lysophosphatidic acid, a Rho stimulator. Assays further revealed that upon myocilin overexpression, the activity of RhoA was diminished, whereas the cAMP level and the protein kinase A (PKA) activity were augmented. Myocilin protein did not affect actin polymerization. The collapse of actin stress fibers and increased trypsin sensitivity from myocilin transfection could be reverted by co-expression of constitutively active RhoA or by treatment with PKA inhibitor H-89. The PKA activity, however, was not modified by co-expression of either constitutively active or dominant negative RhoA. These results demonstrate that myocilin has a de-adhesive activity and triggers signaling events. cAMP/PKA activation and the downstream Rho inhibition are possible mechanisms by which myocilin in overabundance may lead to TM cell or tissue damage.     

Expression of αB-Crystallin in the Peripapillary Glial Cells of the Developing Chick Retina

Peripapillary glial cells (PPGCs) are a peculiar macroglia in avian species, located in the central retina adjacent to the optic nerve head. PPGCs have a similar shape and orientation to Müller cells, which traverse the entire layer of the retina; however, there are differences in protein expression between the two cell types. In the present study, we first demonstrated that PPGCs expressed αB-crystallin, which is not expressed in Müller cells, during retinal development. αB-crystallin was first faintly expressed in PPGCs of the E5 retina, adjacent to the optic nerve head. Further, αB-crystallin was exclusively expressed in PPGCs up to E14. The shape of these cells was bipolar with vitread and ventricular processes. The vitread processes of αB-crystallin+ PPGCs became finer at E18. Double labeling analysis clearly demonstrated that only vimentin+ or GFAP+ astrocytes were located in the optic nerve head and were demarcated from the retina by αB-crystallin+ PPGCs. Furthermore, we determined that αB-crystallin+ PPGCs, with a number of processes, completely wrapped the optic nerve head and were densely located in the junction of the optic nerve head and the retina in a whole mount preparation and in vertical-sectioned retinae. The results of present study, together with reports that retinal astrocytes migrate from the optic nerve head, suggest that PPGCs prevent astrocytes from migrating into the retina in avian species.     

A Role for Myocilin in Receptor-Mediated Endocytosis

Myocilin is a broadly expressed protein that when mutated uniquely causes glaucoma. While no function has been ascribed to explain focal disease, some properties of myocilin are known. Myocilin is a cytoplasmic protein that also localizes to vesicles specifically as part of a large membrane-associated complex with properties similar to the SNARE machinery that function in vesicle fusion. Its role in vesicle dynamics has not been detailed, however myocilin intersects with the endocytic compartment at the level of the multivesicular body. Since internalized GPCRs are sorted in the multivesicular body, we investigated whether myocilin functions in ligand-dependent GPR143 endocytosis. Using recombinant systems we found that the kinetics of myocilin recruitment to biotinylated membrane proteins was similar to that of arrestin-3. We also co-localized myocilin with GPR143 and Arrestin-2 by confocal microscopy. However, wild-type myocilin differed significantly in its association kinetics and co-localization with internalized proteins from mutant myocilin (P370L or T377M). Moreover, we found that myocilin bound to the cytoplasmic tail of GPR143, an interaction mediated by its amino terminal helix-turn-helix domain. Hydrodynamic analyses show that the myocilin-GPR143 protein complex is >158 kD and stable in 500 mM KCl, but not 0.1% SDS. Collectively, data indicate that myocilin is recruited to the membrane compartment, interacting with GPCR proteins during ligand-mediated endocytosis and that GPCR signaling underlies pathology in myocilin glaucoma.     

The role of TGF-β in the pathogenesis of primary open-angle glaucoma

Transforming growth factor-β2 (TGF-β2) is found in increasing amounts in aqueous humor and reactive optic nerve astrocytes of patients with primary open-angle glaucoma (POAG), a major cause of blindness worldwide. The available data strongly indicate that TGF-β2 is a key player contributing to the structural changes in the extracellular matrix (ECM) of the trabecular meshwork and optic nerve head as characteristically seen in POAG. The changes involve an induction in the expression of various ECM molecules and are remarkably similar in trabecular meshwork cells and optic nerve head astrocytes. The ECM changes in the trabecular meshwork most probably play a role in the increase of aqueous humor outflow resistance causing higher intraocular pressure (IOP). In the optic nerve head, TGF-β2-induced changes might contribute to deformation of the optic nerve axons causing impairment of axonal transport and neurotrophic supply and leading to their continuous degeneration. The increase in IOP further adds mechanical stress and strain to optic nerve axons and accelerates degenerative changes. In addition, high IOP might induce the expression of activated TGF-β1 in trabecular meshwork cells and optic nerve head astrocytes; this again might significantly lead to the progress of axonal degeneration. The action of TGF-β2 in POAG is largely mediated through the connective tissue growth factor, whereas the activities of TGF-β1 and -β2 are modulated by the blocking effects of bone morphogenetic protein-4 (BMP-4) and BMP-7, by gremlin that inhibits BMP signaling and by several species of microRNAs.     

Concentration of astrocytic filaments at the retinal optic nerve junction is coincident with the absence of intra-retinal myelination: comparative and developmental evidence

The structure of the lamina cribrosa (LC) and astrocytic density were examined in various species with and without intra-retinal myelination. Sections of optic nerve from various species were stained with Milligan's trichrome or antibodies to glial fibrillary acidic protein, myelin basic protein (MBP) and antibody O4. Marmoset, flying fox, cat, and sheep, which lack intraretinal myelination, were shown to possess a well-developed LC as well as a marked concentration of astrocytic filaments distal to the LC. Rat and mouse, which lack intraretinal myelination, lacked a well-developed LC but exhibited a marked concentration of astrocytic filaments in this region. Rabbit and chicken, which exhibit intraretinal myelination, lacked both a well-developed LC and a concentration of astrocytes at the retinal optic nerve junction (ROJ). A marked concentration of astrocytes at the ROJ of human fetuses was also apparent at 13 weeks of gestation, prior to myelination of the optic nerve; in contrast, the LC was not fully developed even at birth. This concentration of astrocytes was located distal to O4 and MBP immunoreactivity in human optic nerve, and coincided with the site of initial myelination of ganglion cell axons in marmoset and rat. Myelination proceeded from the chiasm towards the retinal end of the human optic nerve. Moreover, the outer limit of oligodendrocyte precursor cells (OPC) migration into the rabbit retina was restricted by the outer limit of astrocyte spread. These observations indicate that a concentration of astrocytic filaments at the ROJ is coincident with the absence of intraretinal myelination. Differential expression of tenascin-C by astrocytes at the ROJ appears to contribute to the molecular barrier to OPC migration (see Bartsch et al., 1994), while expression of the homedomain protein Vax 1 by glial cells at the optic nerve head appears to inhibit migration of retinal pigment epithelial cells into the optic nerve (see Bertuzzi et al., 1999). These observations combined with our present comparative and developmental data lead us to suggest that the astrocytes at the ROJ serve to regulate cellular traffic into and out of the retina.     

Myocilin promotes substrate adhesion,spreading and formation of focal contacts in podocytes and mesangial cells

Myocilin, a secreted glycoprotein of the olfactomedin family, is constitutively expressed in podocytes of the rat kidney and induced in mesangial cells during mesangioproliferative glomerulonephritis. As myocilin has been found to be associated with fibrillar components of the extracellular matrix, and adhesive properties have been shown for other members of the olfactomedin family, we hypothesized that myocilin might play a role in cell-matrix interactions in the glomerulus. To elucidate functional properties of myocilin, recombinant myocilin was expressed in 293 EBNA cells and purified by Ni-chelate and heparin chromatography. Culture plates were coated with myocilin, and primary rat mesangial cells and cells from an immortal murine podocyte cell line were seeded onto the plates in serum free conditions. Both cell types showed concentration-dependant attachment to myocilin, an effect that was statistically significant and could be blocked with specific antibodies. When compared to equal amounts of fibronectin or collagen 1, myocilin was less effective in promoting substrate adhesion. Synergistic effects in substrate adhesion were observed when myocilin was added to low concentrations of fibronectin. Twenty-five percent of cells that had attached to myocilin substrates showed spreading and expressed focal contacts which were labeled by vinculin/phalloidin staining. Comparable findings were observed when human or murine trabecular meshwork cells were seeded on myocilin substrates. Adhesive properties of myocilin required multimer formation, and were not observed when culture plates were coated with a C-terminal fragment of myocilin, containing the olfactomedin domain. We conclude that myocilin promotes substrate adhesion of podocytes and mesangial cells, and might contribute to cell-matrix adhesion of both cell types in vivo.     

Comparison of the glial investment of normal and regenerating fiber bundles in the optic nerve and optic tectum of the goldfish and the Crucian carp

Summary The architecture of normal and regenerating nerve fiber bundles in the optic nerve of the goldfish and the Crucian carp was compared to that of the axonal fascicles in the optic tectum of these teleost species with the use of ultrathin sections and freeze-fracture replicas. The fascicles in the optic nerve are clearly demarcated by astrocytic processes, in contrast to the fascicles in the tectum. No astrocytes could be identified in the tectum; in this region processes of astrocytes or of radial glial cells do not form channeling structures reminiscent of those in the optic nerve. Furthermore, tectal blood vessels lack complete investments of glial processes. It can be assumed that at least in lower vertebrates a framework of astrocytic processes might be important for growth of optic fibers over large distances, i.e., from the eye to the tectum, but may be dispensable in the target region itself.     

The Glaucoma-associated Olfactomedin Domain of Myocilin Is a Novel Calcium Binding Protein

Myocilin is a protein found in the trabecular meshwork extracellular matrix tissue of the eye that plays a role in regulating intraocular pressure. Both wild-type and certain myocilin variants containing mutations in the olfactomedin (OLF) domain are linked to the optic neuropathy glaucoma. Because calcium ions are important biological cofactors that play numerous roles in extracellular matrix proteins, we examined the calcium binding properties of the myocilin OLF domain (myoc-OLF). Our study reveals an unprecedented high affinity calcium binding site within myoc-OLF. The calcium ion remains bound to wild-type OLF at neutral and acidic pH. A glaucoma-causing OLF variant, myoc-OLF(D380A), is calcium-depleted. Key differences in secondary and tertiary structure between myoc-OLF(D380A) and wild-type myoc-OLF, as well as limited access to chelators, indicate that the calcium binding site is largely buried in the interior of the protein. Analysis of six conserved aspartate or glutamate residues and an additional 18 disease-causing variants revealed two other candidate residues that may be involved in calcium coordination. Our finding expands our knowledge of calcium binding in extracellular matrix proteins; provides new clues into domain structure, function, and pathogenesis for myocilin; and offers insights into highly conserved, biomedically relevant OLF domains.     

Mitochondrial association of myocilin, product of a glaucoma gene, in human trabecular meshwork cells

The trabecular meshwork (TM), an ocular tissue next to the cornea, is a major site for regulation of the aqueous humor outflow. Malfunctioning of this tissue is believed to be responsible for development of glaucoma, a major blinding disease. Myocilin is a gene directly linked to the most common form of glaucoma. Its protein product has been localized to both intra- and extra-cellular sites in TM cells. This study was to investigate the association of myocilin with mitochondria in TM cells. In vitro mitochondrial import assays showed that myocilin was imported to the TM mitochondria, targeting to mitochondrial membranes and/or the intermembrane space. The targeting was mediated mostly via the amino-terminal region of myocilin. When myocilin expression was induced either by treatment with dexamethasone or transfection with a myocilin construct, the mitochondrial membrane potential in TM cells, as assessed by JC-1 staining, was lowered. Subcellular fractionation and Western blot analyses confirmed that a portion of myocilin sedimented with the mitochondrial fractions. Upon anti-Fas treatment to provoke apoptosis, an increase of myocilin distribution in cytosolic fraction was observed, suggesting that myocilin was partially released from mitochondrial compartments. These results confirmed the association of myocilin with TM cell mitochondria and indicated that myocilin may have a proapoptotic role in TM cells.     

Proteomics analyses of human optic nerve head astrocytes following biomechanical strain

We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor β1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin.     

The astrocytes in the retina and optic nerve head of mammals: A special glia for the ganglion cell axons

Summary The neuroglia in the retina and the intraocular portion of the optic nerve of the monkey and cat has been examined by light and electron microscopy. In the retina two types of macroglial cells can be distinguished: 1) Müller cells, and 2) astrocytes. The bipolar radial glial cells of Müller penetrate the entire thickness of the retina and their basal processes align in the nerve fibre layer to form septa that fasciculate the axons of the ganglion cells. In contrast to the Müller cells, the retinal astrocytes are not homogeneously distributed throughout the retina; their number correlates with the thickness of the nerve fibre layer. The processes of the astrocytes are confined to the ganglion cell layer and to the nerve fibre layer. In the latter, the astrocytic processes run parallel to and between the axons of a given nerve fibre bundle. According to cytological criteria, the retinal astrocytes are protoplasmic. In the intraocular portion of the optic nerve, however, the astrocytes are fibrous and their processes run perpendicular to the axon bundles of the prelaminar portion of the optic nerve. Thus, because of their intimate morphological relationship to axons of the nerve fibre layer and the intraocular portion of the optic nerve, the astrocytes in the eye of the monkey and the cat may be considered as a special glia for the axons of ganglion cells.     

Overexpression of myocilin in cultured human trabecular meshwork cells

The trabecular meshwork, a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of the trabecular meshwork is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. Its expression is known to be upregulated by glucocorticoids in trabecular meshwork cells and the altered myocilin level may be the culprit for glaucomatous conditions such as corticosteroid-induced glaucoma. In this study, we examined the influence of myocilin overexpression on the adhesion, spreading, migration, phagocytosis, and apoptosis of human trabecular meshwork cells in culture. When the myocilin expression was increased by 3- to 4-fold, the transfectants showed a dramatic loss of actin stress fibers and focal adhesions. Cell adhesion to fibronectin and spreading were also compromised. Myocilin thus appeared to have a de-adhesive activity, similar to that reported extensively with matricellular proteins. The transfected cells in addition displayed an increased sensitivity to apoptosis. These results demonstrate that overexpression of myocilin renders trabecular meshwork cells in a de-adhesive and vulnerable state. This vulnerability may be the basis for pathologic consequences in subtypes of glaucoma.