Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues.

PURPOSE: To examine the intracellular and extracellular expression of myocilin in the human and primate trabecular meshwork (TM) in the presence and absence of glucocorticoids. METHODS: Myocilin expression was examined in cultured human TM cells by Northern blot analysis and myocilin antibody-mediated immunoprecipitation. Myocilin expression was quantified using high-resolution two-dimensional polyacrylamide gel electrophoresis of radiolabeled proteins from human TM cells, TM tissue explants, and perfused human anterior segments cultured with and without dexamethasone (DEX) for 14 to 21 days, as well as TM tissue from pigtailed monkeys treated orally for 1 year with cortisone acetate. Immunofluorescence with anti-myocilin antibodies was used to localize cellular and extracellular expression of myocilin in cultured human TM cells. RESULTS: Glucocorticoid treatment caused a significant induction of myocilin mRNA, a tetrad of cell-associated proteins, and 8 to 20 secreted proteins (molecular mass [M(r)] 56 and 59 kDa and isoelectric point [pI] 5.2 and 5.3) in some, but not all the cultured human TM cells and explanted tissues. Western immunoblot analysis using anti-myocilin peptide antibodies identified these proteins as encoded by the MYOC gene. There was significant induction of the myocilin proteins in three perfusion-cultured human eyes, in which DEX-induced elevated intraocular pressure developed. Monkeys treated 1 year with cortisol acetate showed steroid glaucoma-like morphologic changes in the TM that correlated with the induction of myocilin in the TM. Immunofluorescence analysis of cultured TM cells localized myocilin intracellularly in discrete perinuclear and cytoplasmic vesicular deposits as well as extracellularly on the cell surface associated with the extracellular matrix. In several DEX-treated TM cell lines, there were significant levels of myocilin secreted into the media. Enzymatic deglycosylation of proteins in the TM media converted the higher molecular weight isoforms of myocilin (approximately 57 kDa) to the lower molecular weight isoforms ( approximately 55 kDa). CONCLUSIONS: Although the function of myocilin is unknown, induction of these TM proteins was found in eyes in which glucocorticoid-induced ocular hypertension developed. Therefore, myocilin may play an important pathogenic role in ocular hypertension in addition to its role in certain forms of POAG.

Analysis of UVA-related alterations upon aging of eye lens proteins by mini two-dimensional polyacrylamide gel electrophoresis.

This study is a first approach to identify UVA-related alterations in situ of bovine eye lens proteins from the water-soluble and urea-soluble fractions upon aging. The fractions were obtained from irradiated long-term organ culture lenses and analyzed by mini two-dimensional gel electrophoresis. This micropreparative method followed by computer analysis allows high resolution and separation of microgram quantities of proteins and to detect spots which arose as a consequence of irradiation. To facilitate the analysis we first separated the water-soluble fraction into the major crystallin classes by gel filtration. Moreover, we immunoblotted the gel of the urea-soluble fraction with a specific antibody against the intermediate filament protein vimentin. Upon irradiation of young and adult lenses, alphaA-crystallin and vimentin showed obvious modifications. During aging the susceptibility to irradiation increased when vimentin started to degrade, whereas deamidation of alphaA-crystallin seems to occur.

The similarity of protein expression in trabecular meshwork and lamina cribrosa: implications for glaucoma.

The purpose of the present investigation was to compare protein expression in various ocular cells and tissues including the human trabecular meshwork (TM) and the lamina cribrosa (LC). To conduct the comparisons, we primarily utilized autofluorography of one-dimensional (1D) and high resolution, two-dimensional (2D) polyacrylamide gels of proteins from radiolabelled tissues and cultured cells. Results from the investigations indicated that patterns of protein expression from TM and LC were the most similar among the ocular cells and tissues compared.Specifically, these autofluorographic ' fingerprints' indicated that proteins in TM and LC cultured cells and tissue were exceptionally similar (a) in band position and intensity (1D gels) and (b) in spot congruence (2D gels) as compared to other ocular cells and tissues. We conclude that the TM and the LC, two ocular tissues intimately linked to the pathogenesis of primary open-angle glaucoma, display remarkable similarity in protein expression. This finding may have implications for the molecular etiology of glaucoma.

The use of proteomics in ophthalmic research.

The goal of molecular ophthalmology is the early detection and therapeutic treatment of eye disease. Genomic technologies have profoundly enhanced the discovery of ocular disease candidate genes. Proteomics, the protein cognate of genomic technology, offers a means to monitor changes in the expression of a given ocular protein(s) and its post-translational modification, identify novel therapeutic targets and evaluate pharmacological effects on a given metabolic pathway. Using both tissue and cultured cells, numerous laboratories have begun to catalogue changes in ocular protein expression in normal, diseased and ageing subjects. Herein, we review published proteomic literature in the broad context of ophthalmic diseases involving various tissues of the eye.

The effect of dexamethasone on integrin and laminin expression in cultured human trabecular meshwork cells.

Glucocorticoid treatment in vivo can produce a glaucoma similar in many ways to POAG. Treatment of trabecular meshwork cells in culture with dexamethasone allows the study of biochemical aspects of this disease process. The effects of dexamethasone on the expression of integrins and laminin in both normal and glaucomatous cultured human trabecular meshwork cells were evaluated. Human trabecular meshwork cell lines were cultured for 18 days in the presence or absence of 10(-7) m dexamethasone. Radioimmunoprecipitation was used to determine the relative expression of five alphaintegrin subunits. Laminin expression was evaluated with Western blots. Laminin was increased in all cell lines following dexamethasone treatment. alpha2, alpha5 and alphaV integrin chains showed consistent dexamethasone-induced changes in expression, while alpha3 and alpha4 subunits did not. There were no differences in the expression patterns for any of these integrin subunits between normal and glaucomatous cell lines. Increased laminin deposition as seen in this study with dexamethasone treatment may be partially responsible for the decreased outflow facility seen in both steroid-induced glaucoma and in POAG.

Towards a human crystallin map. Two-dimensional gel electrophoresis and computer analysis of water-soluble crystallins from normal and cataractous human lenses.

This paper describes a first approach to establish a master data base of human lens crystallins obtained by computer analysis of standardized two-dimensional lenticular protein patterns. To facilitate the eventual identification of the spots, the major crystallins have been separated into alpha-, beta H-, beta L- and gamma-crystallin fractions by gel filtration. The authors encourage colleague investigators to collaborate in a common effort in order to arrive eventually at a two-dimensional gel data base of all lenticular proteins.

Presence of functional type B natriuretic peptide receptor in human ocular cells.

PURPOSE: To study the effects of natriuretic peptides on cyclic guanosine monophosphate (cGMP) production and calcium mobilization in cultured human ocular cells. METHODS: Cultured simian virus 40-transformed (HTM-3) and nontransformed (HTM-16) human trabecular meshwork (TM) cells and nontransformed human ciliary muscle (CM) cells were used. Accumulation of cGMP in cells lysate was measured by radioimmunoassay. Intracellular calcium concentration was measured by microscope-based ratiofluorometry. RESULTS: Both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) increased the accumulation of cGMP in HTM-3, HTM-16, and CM cells. In the nontransformed TM cells, CNP was five times more efficacious (maximal effect of CNP was 497% +/- 44% that of ANP) and 10 times more potent than ANP (ANP, log [EC50] = -6.99 +/- 0.08; CNP, log [EC50] = -7.96 +/- 0.20). Similar results were seen in HTM-3 and CM cells. Under the assay conditions used, the peptides increased only the production of cGMP without changing its degradation rate. The peptide-induced increase of cGMP in the TM and CM cells correlated with suppression of carbachol-induced calcium mobilization in the cell. CONCLUSIONS: It is known that CNP, but not ANP, selectively activates the guanylyl cyclase associated with the type B natriuretic peptide receptor (NPR-B). Thus, the data suggest that NPR-B is the primary functional NPR in the TM and CM cells. The effects on cGMP and calcium produced by the activation of this receptor are expected to alter TM and CM contractility and may affect aqueous humor hydrodynamics and intraocular pressure.

Preliminary characterization of a transformed cell strain derived from human trabecular meshwork.

Cells isolated from the trabecular meshwork (TM) of a male glaucoma patient were transformed by transfection with an origin defective mutant of SV40 virus. Transformation dramatically increased the growth rate of these cells (designated HTM-3 cells), allowing biochemical and pharmacological characterization. The HTM-3 cells had cytoskeletal components that were reported to be present in TM tissue and non-transformed TM cells. Vimentin, tubulin and smooth muscle specific alpha-actin, but not desmin, were localized in these cells by immunocytochemistry. The extracellular matrix components collagen types I, III and IV, fibronectin and laminin were found in HTM-3 cells as well as their non-transformed parental cells. As predicted, the protein profile of the HTM-3 cells revealed by two-dimensional gel electrophoresis was different from that of the non-transformed cells, probably due to the enhanced growth characteristics of these cells. Furthermore, HTM-3 cells had various intracellular second messenger systems that responded to pharmacological agents. Forskolin, prostaglandin E2, beta-adrenergic and adenosine A2 agonists stimulated the adenylyl cyclase in these cells, whereas muscarinic, serotonergic, dopaminergic and other agonists were ineffective. Sodium nitroprusside increased the intracellular concentration of cGMP, demonstrating the presence of a functional guanylyl cyclase. Phospholipase C activity in these cells was also detected. Muscarinic agonists, histamine and bradykinin, but not adrenergic, serotonergic agonists or prostaglandins, increased phosphoinositide turnover. These drug responses of HTM-3 cells agree with published data on primary TM cells and TM tissues, suggesting that the transformed cells may be a valid substitute for certain pharmacological studies of TM.

The effects of dexamethasone on fibronectin expression in cultured human trabecular meshwork cells.

Topical administration of glucocorticoids to the eye can lead to the development of ocular hypertension. This increase in intraocular pressure is caused by the heightened resistance to flow of aqueous humor from the eye, presumably at the trabecular meshwork (TM). This study reports the effects of dexamethasone (DEX) on the expression of the extracellular matrix protein fibronectin (FN) in cultured human TM cells (HTM). The expression of FN was evaluated in four HTM cell strains by epifluorescence microscopy and immunoblotting and autofluorography of electrophoretically separated cell proteins. There was a heterogeneous response of the four cell strains tested. Treatment of cell strain HTM4 with DEX (10(-7) mol/l) for 17 d caused an approximate doubling of cell-associated and secreted FN. This DEX-induced increase in FN expression was progressive after the first 7 d of treatment and was blocked partially with a glucocorticoid antagonist, cortexolone. By contrast, DEX treatment induced an intermediate 50-60% increase in FN expression in cell strains HTM10 and HTM2; in HTM6, FN was unchanged after exposure to the glucocorticoid. This model system may be useful to examine molecular changes associated with corticosteroid-induced ocular hypertension and evaluate glaucomatous changes in the TM because increased FN deposition occurs in the aqueous humor outflow pathway of patients with open-angle glaucoma.

Length, mass, and denaturation of double-stranded RNA molecules compared with DNA.

The contour lengths of linear, double-stranded (ds) RNAs from mycovirus PcV and Pseudomonas bacteriophage phi 6 have been measured with samples prepared for the electron microscope from 0.05 to 0.5 M NH4Cl solutions. A linear dependence of contour length on the logarithm of ionic strength was found and compared with that of dsDNA (pBR322, linearized and open-circular forms). Conditions for molecular weight determinations of any natural dsRNA by electron microscopy have been established, and the method has been calibrated with phi 6 dsRNA of known nucleotide sequence. The results imply that dsRNA in 0.20 M NH4Cl solution has a rise per basepair of 0.271 nm, which is shorter than that in the A-conformation (4%) and in the A'-conformation (10%). The thermal behavior of dsRNA in terms of melting temperature and exhibition of fine structure of melting curves was found to be generally similar to that of dsDNA, as expected from the literature. Folding of dsRNA in ethanolic solution was similar to that of dsDNA. However, in contrast to dsDNA, coiled coils could not be induced by ethanol, which is consistent with dsRNA being stiffer than dsDNA. Concerning dsDNA, the results show that a contraction in rise per basepair by 0.1 nm is coupled with an increase in the winding angle between basepairs by 0.47 degrees, as qualitatively predicted by polyelectrolyte theory.

CD of homopolymer DNA-RNA hybrid duplexes and triplexes containing A-T or A-U base pairs.

CD spectra and difference-CD spectra of (a) two DNA X RNA hybrid duplexes (poly[r(A) X d(U)] and poly[r(A) X d(T)]) and (b) three hybrid triplexes (poly-[d(T) X r(A) X d(T)], poly[r(U) X d(A) X r(U)], and poly[r(T) X d(A) X r(T)]) were obtained and compared with CD spectra of six A X U- and A X T-containing duplex and triplex RNAs and DNAs. We found that the CD spectra of the homopolymer duplexes above 260 nm were correlated with the type of base pair present (A-U or A-T) and could be interpreted as the sum of the CD contributions of the single strands plus a contribution due to base pairing. The spectra of the duplexes below 235 nm were related to the polypurine strands present (poly-[r(A)] or poly[d(A)]). We interpret the CD intensity in the intermediate 255-235 nm region of these spectra to be mainly due to stacking of the constituent polypurine strands. Three of the five hybrids (poly[r(A) X d(U)], poly[r(A) X d(T)], and poly[d(T) X r(A) X d(T)]) were found to have heteronomous conformations, while poly[r(U) X d(A) X r(U)] was found to be the most A-like and poly[r(T) X d(A) X r(T)], the least A-like.

Electron microscopy of bacteriophage phi 6 nucleocapsid: two-dimensional image analysis.

Electron micrographs of negatively stained nucleocapsids isolated from intact, wild-type phi 6 bacteriophage revealed three distinct morphological forms. Two-dimensional analysis of electron micrographs of two of these forms and image averaging of all forms are consistent with a dodecahedral structure embodied in the phi 6 nucleocapsid.