CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix.

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients' features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.

A renal-like organic anion transport system in the ciliary epithelium of the bovine and human eye.

The purpose of this study was to determine the direction of organic anion (OA) transport across the ciliary body and the transport proteins that may contribute. Transport of several OAs across the bovine ciliary body was examined using ciliary body sections mounted in Ussing chambers and a perfused eye preparation. Microarray, reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry were used to examine OA transporter expression in human ocular tissues. Microarray analysis showed that many OA transporters common to other barrier epithelia are expressed in ocular tissues. mRNA (RT-PCR) and protein (immunoblotting) for OAT1, OAT3, NaDC3, and MRP4 were detected in extracts of the human ciliary body from several donors. OAT1 and OAT3 localized to basolateral membranes of nonpigmented epithelial cells and MRP4 to basolateral membranes of pigmented cells in the human eye. Para-aminohippurate (PAH) and estrone-3-sulfate transport across the bovine ciliary body in the Ussing chambers was greater in the aqueous humor-to-blood direction than in the blood-to-aqueous humor direction, and active. There was little net directional movement of cidofovir. Probenecid (0.1 mM) or novobiocin (0.1 mM) added to the aqueous humor side of the tissue, or MK571 (5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid; 0.1 mM) added to the blood side significantly reduced net active PAH transport. The rate of 6-carboxyfluorescein elimination from the aqueous humor of the perfused eye was reduced 80% when novobiocin (0.1 mM) was present in the aqueous humor. These data indicate that the ciliary body expresses a variety of OA transporters, including those common to the kidney. They are likely involved in clearing potentially harmful endobiotic and xenobiotic OAs from the eye.

The blood-aqueous barrier in health and disease.

The blood-aqueous barrier of the eye is composed by tight junctions in the ciliary process nonpigmented epithelium, the endothelial cells in the iris vasculature, and the inner wall endothelium of Schlemm's canal. Tight junctions are gatekeepers of the paracellular transport limiting the selective diffusion of ions and small solutes through the space between neighboring cells. Tight junctions (ie, junctional adhesion molecules, claudins, occludins, zonula occludens, cingulin) are part of the apical junctional complex that also includes the adherens junctions (ie, cadherin-catenin and nectin-afadin complexes) and the gap junctions (ie, connexins). These junctional complexes respond rapidly to pharmacologic agents and physiological changes. Barrier dysfunction can contribute to the pathophysiology of inflammatory ocular diseases in a passive way by the vascular leakage of blood-borne molecules and inflammatory cells into the anterior segment of the eye.

Characterization of vitamin D production by human ocular barrier cells.

PURPOSE: Vitamin D3 is a secosteroid mainly synthesized from the conversion of the skin precursor 7-dehydrocholesterol (7DHC) to vitamin D3 by ultraviolet (UV) B sunlight. Extrarenal synthesis of vitamin D3 has been reported in many tissues and cells, including barrier sites. This study characterizes the expression of components of vitamin D3 signaling in human ocular barrier cells. METHODS: Primary human scleral fibroblasts (HSF), human corneal endothelial (HCEC-12), nonpigmented ciliary body epithelial (ODM-2), and adult retinal pigment epithelial (ARPE-19) cell lines were analyzed for the expression of vitamin D receptor (VDR), the vitamin D3 activating enzymes 1α-hydroxylase (CYP27B1), 25-hydroxylases (CYP27A1 and CYP2R1), the vitamin D3 inactivating enzyme 24-hydroxylase (CYP24A1), and the endocytic receptors cubilin and megalin using a combination of RT-PCR, immunocytochemistry, and enzyme immunoassay (EIA). RESULTS: The HSF, HCEC-12, ODM-2, and ARPE-19 express mRNA and protein for all vitamin D3 synthesizing and metabolizing components. The cell types tested, except HSF, are able to convert inactive 25-hydroxyvitamin D3 (25[OH]D3) into active 1,25-hydroxyvitamin D3 (1,25[OH]2D3). CONCLUSIONS: This novel study demonstrated that ocular barrier epithelial cells express the machinery for vitamin D3 and can produce 1,25(OH)2D3. We suggest that vitamin D3 might have a role in immune regulation and barrier function in ocular barrier epithelial cells.

The stoichiometric transition from Zn6Cu1-metallothionein to Zn7-metallothionein underlies the up-regulation of metallothionein (MT) expression: quantitative analysis of MT-metal load in eye cells.

We examined the profiling of gene expression of metallothioneins (MTs) in human tissues from cadaver eyes with microarray-based analysis. All MT1 isoforms, with the exception of MT1B, were abundantly expressed in lens and corneal tissue. Along with MT1B, MT4 was not detected in any tissues. Antibodies to MT1/2 labeled the corneal epithelial and endothelial cells, whereas MT3 label the retinal ganglion cells. We studied the effects of zinc and cytokines on the gene expression of MT isoforms in a corneal epithelial cell line (HCEsv). Zinc exerted an up-regulation of the expression of MT isoforms, and this effect was further potentiated in the presence of IL1α or TNFα. Zinc also elicited a strong down-regulation of the expression of inflammatory cytokines, and this effect was blocked in the presence of TNFα or IL1α. The concentration of MTs, bound zinc, and the metal stoichiometry of MTs in cultured HCEsv were determined by mass spectrometry. The total concentration of MTs was 0.24 ± 0.03 µM and, after 24 h of zinc exposure, increased to 0.96 ± 0.01 µM. The combination of zinc and IL1α further enhanced the level of MTs to 1.13 ± 0.03 µM. The average metal stoichiometry of MTs was Zn(6)Cu(1)-MT, and after exposure to the different treatments, it changed to Zn(7)-MT. Actinomycin D blocked transcription, and cycloheximide attenuated synthesis of MTs in the presence or absence of zinc, suggesting transcriptional regulation. Overall the data provide molecular and analytical evidence on the interplay between zinc, MTs, and proinflammatory cytokines in HCEsv cells, with potential implications on cell-based inflammatory eye diseases.

WDR36 and P53 gene variants and susceptibility to primary open-angle glaucoma: analysis of gene-gene interactions.

PURPOSE: To investigate the role of WDR36 and P53 sequence variations in POAG susceptibility. METHODS: The authors performed a case-control genetic association study in 268 unrelated Spanish patients (POAG1) and 380 control subjects matched for sex, age, and ethnicity. WDR36 sequence variations were screened by either direct DNA sequencing or denaturing high-performance liquid chromatography. P53 polymorphisms p.R72P and c.97-147ins16bp were analyzed by single-nucleotide polymorphism (SNP) genotyping and PCR, respectively. Positive SNP and haplotype associations were reanalyzed in a second sample of 211 patients and in combined cases (n = 479). RESULTS: The authors identified almost 50 WDR36 sequence variations, of which approximately two-thirds were rare and one-third were polymorphisms. Approximately half the variants were novel. Eight patients (2.9%) carried rare mutations that were not identified in the control group (P = 0.001). Six Tag SNPs were expected to be structured in three common haplotypes. Haplotype H2 was consistently associated with the disease (P = 0.0024 in combined cases). According to a dominant model, genotypes containing allele P of the P53 p.R72P SNP slightly increased glaucoma risk. Glaucoma susceptibility associated with different WDR36 genotypes also increased significantly in combination with the P53 RP risk genotype, indicating the existence of a genetic interaction. For instance, the OR of the H2 diplotype estimated for POAG1 and combined cases rose approximately 1.6 times in the two-locus genotype H2/RP. CONCLUSIONS: Rare WDR36 variants and the P53 p.R72P polymorphism behaved as moderate glaucoma risk factors in Spanish patients. The authors provide evidence for a genetic interaction between WDR36 and P53 variants in POAG susceptibility, although this finding must be confirmed in other populations.

Interaction of recombinant myocilin with the matricellular protein SPARC: functional implications.

PURPOSE: Myocilin is an extracellular glycoprotein with unknown function that is associated with glaucoma. Calpain II cleaves recombinant myocilin within the linker region of the protein, releasing the C-terminal olfactomedin domain from the N-terminal domain. The authors previously reported that myocilin interacts with the C-terminal region of hevin, a secretory glycoprotein belonging to the SPARC family of matricellular proteins. This study aims to investigate the interaction of myocilin with SPARC. METHODS: Protein-protein interactions were evaluated by the yeast two-hybrid system. The positive interactions were confirmed by solid-phase binding assays using Ni-chelating HPLC purified recombinant proteins and coexpression of recombinant proteins in HEK-293T cells. Coexpression of myocilin, SPARC, and hevin in ocular tissues was identified by immunoflorescence microscopy, Western blot, and array-based gene profiling. RESULTS: Yeast two-hybrid analyses showed that myocilin interacted with the highly conserved C-terminal extracellular calcium binding (EC) domain within SPARC and hevin. Solid-phase binding assays confirmed these interactions and showed that both myocilin and its C-terminal olfactomedin fragment interacted noncovalently with SPARC and a peptide containing the EC domain of SPARC. Full-length myocilin interacted with higher affinity with SPARC and its EC domain than the myocilin C-terminal fragment. Coexpression of the two recombinant proteins in HEK-293T cells also indicated their intracellular interaction. CONCLUSIONS: Recombinant myocilin and SPARC interact through their C-terminal domains. The data suggest that the proteolytic processing of myocilin modulates this interaction as well as the interactions of myocilin with other extracellular matrix and matricellular proteins, further supporting a functional role for this proteolytic cleavage.

Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris.

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.

Molecular analysis of neurolysin expression in the rat and bovine ciliary body.

PURPOSE: This paper deals with the capability of the ciliary epithelium to express neurolysin, involved in the inactivation of numerous neuropeptides. METHODS: Total RNAs from ciliary body (CB) were processed for RT-PCR, and the amplification products were sequenced. The whole-protein extracts of CBs were analyzed using the Western blot. The CBs were processed for neurolysin immunolocalization. RESULTS: The RT-PCR detected the presence of neurolysin mRNA in the ciliary body. The Western blot assays demonstrated immunochemical cross-reactivity with neurolysin. The immunoreactivity to neurolysin was observed in ciliary epithelium. CONCLUSIONS: These results indicate that the ciliary epithelium expresses neurolysin.

New perspectives in aqueous humor secretion and in glaucoma: the ciliary body as a multifunctional neuroendocrine gland.

The discovery in the human ocular ciliary body of glaucoma-associated genes (i.e., MYOC, CYP1B1), neuroendocrine processing enzymes, neuroendocrine peptides, steroid-converting enzymes, glutamate transporters, glutamate-metabolizing enzymes, and anti-angiogenic factors requires a reevaluation of its function on aqueous humor secretion, intraocular pressure and its role in glaucoma. The ciliary body should be considered as a multifunctional and interactive tissue. The intrinsic hypotensive and/or hypertensive biological activities of many of the endocrine peptides released by the ciliary epithelium are best explained within the context of a neuroendocrine system, linking the inflow and the outflow of aqueous humor. This interpretation is consistent with physiological and genetic studies indicating that changes altering the inflow affects intraocular pressure. In the proposed endocrine system, regulatory peptides secreted by the ciliary epithelium may subserve multiple functions in the following: inflow and outflow pathways of aqueous humor, ciliary blood flow, the immune privilege status of the anterior segment and the diurnal circadian rhythms of aqueous humor secretion and intraocular pressure. These previously unsuspected and challenging functions of the ciliary epithelium should be considered when assessing the multifactorial events which lead to the pathophysiology of glaucoma affecting the outflow pathways of aqueous humor. This review highlights published, and ongoing studies on authors' labs supporting neuroendocrine, steroidogenic and glutamatergic features of the ciliary epithelium and the endocrine communication between the inflow and outflow pathways of aqueous humor. We also discuss how glaucoma-associated genes expressed in the ciliary body and their mutant proteins could influence intraocular pressure, contributing to the development of glaucoma.

Heterozygous CYP1B1 gene mutations in Spanish patients with primary open-angle glaucoma.

PURPOSE: To investigate CYP1B1 gene mutations in Spanish patients with ocular hypertension (OHT) or primary open angle glaucoma (POAG). METHODS: The two coding exons of CYP1B1 were screened for sequence alterations by direct PCR DNA sequencing in 37 and 82 unrelated Spanish subjects diagnosed with OHT and POAG, respectively. As a control we used a group of 93 subjects from whom OHT or glaucoma were ruled out. RESULTS: We found three different predicted amino acid substitutions (Ala189Pro, Ala330Ser, and Ala443Gly) in three (8.1%) OHT subjects, and seven different mutations (Ser28Trp, Gly61Glu, Tyr81Asn, Gln144His, Arg145Trp, Glu229Lys, and Val409Phe) in nine (10.9%) glaucoma patients. These sequence variations showed higher frequencies in cases than in controls (as recently reported in French patients). They are predicted to produce a significant change in the amino acid sequence and affect conserved regions of the protein. All these missense mutations were found as heterozygots. In addition, four of them have been previously found in PCG and/or POAG patients, whereas the other six mutations (Ser28Trp, Gln144His, Arg145Trp, Ala189Pro, Ala330Ser, and Val409Phe) have not been previously described. Clinically, these mutations are associated with an age at diagnosis ranging from 12 to 58 years (mean 34.3 years) and from 48 to 77 years (mean 59.9 years) among OHT and glaucoma patients, respectively. CONCLUSIONS: Heterozygous CYP1B1 mutations could confer increased susceptibility to the development of POAG in the Spanish population.

Somatostatin modulates PI3K-Akt, eNOS and NHE activity in the ciliary epithelium.

Somatostatin (SST) is a biologically active peptide produced in neuroendocrine cells. In the present study, we provide evidence of pro-SST and SST receptor (SSTR1 and 2A) mRNA expression in ocular ciliary epithelium (CE). SST or SST-like immunoreactivity was detected by radioimmunoassay in tissue extract from ciliary processes and in aqueous humor. The distinct immunolabeling of CE with SST and proprotein convertases PC1 and PC2 antibodies suggested a tissue and cell-specific processing of pro-SST. SST (10(-8) to 10(-4)M) added exogenously to the CE, elicited the following effects: (i) a dose-dependent attenuation of Na+/H+-exchanger (NHE) activity; (ii) up to a two-fold increase phosphorylation of p-Akt-Ser473 and of p-eNOS-Ser617, and (iii) lack of response on intracellular cyclic GMP production. LY294002, a PI3K-inhibitor, blocked SST-induced p-Akt-Ser473 and partially p-eNOS-Ser617, however, it did not reverse SST-induced NHE attenuation. Collectively, these results suggested involvement of SST in multiple intracellular signaling pathways in the CE.

The cross-species A3 adenosine-receptor antagonist MRS 1292 inhibits adenosine-triggered human nonpigmented ciliary epithelial cell fluid release and reduces mouse intraocular pressure.

PURPOSE: Antagonists to A3 adenosine receptors (ARs) lower mouse intraocular pressure (IOP), but extension to humans is limited by species variability. We tested whether the specific A3AR antagonist MRS 1292, designed to cross species, mimicks the effects of other A3AR antagonists on cultured human nonpigmented ciliary epithelial (NPE) cells and mouse IOP. METHODS: NPE cell volume was monitored by electronic cell sorting. Mouse IOP was measured with the Servo-Null Micropipette System. RESULTS: Adenosine triggered A3AR-mediated shrinkage of human NPE cells. Shrinkage was blocked by MRS 1292 (IC50 = 42 +/- 11 nM, p < 0.01) and by another A3AR antagonist effective in this system, MRS 1191. Topical application of the A3AR agonist IB-MECA increased mouse IOP. MRS 1292 reduced IOP by 4.0 +/- 0.8 mmHg at 25-microM droplet concentration (n = 10, p < 0.005). CONCLUSIONS: MRS 1292 inhibits A3AR-mediated shrinkage of human NPE cells and reduces mouse IOP, consistent with its putative action as a cross-species A3 antagonist.

Selective upregulation of the A3 adenosine receptor in eyes with pseudoexfoliation syndrome and glaucoma.

PURPOSE: Adenosine is increasingly released in metabolic stress conditions, like hypoxia or ischemia, and regulates many physiologic processes, such as aqueous humor secretion and intraocular pressure, via activation of four adenosine receptors. In the current study, the role of the adenosine system in the pathophysiology of pseudoexfoliation (PEX) syndrome, which is typically associated with anterior chamber hypoxia and elevated intraocular pressure, was examined. METHODS: RT-PCR, Northern hybridization, in situ hybridization, and immunohistochemistry were applied to analyze the mRNA and protein expression of the adenosine receptor subtypes A1, A2A, A2B, and A3 in anterior segment tissues of PEX eyes, without and with glaucoma, in comparison to eyes with primary open-angle or angle-closure glaucoma and normal control eyes. Real-time PCR was used to study the effect of hypoxia and oxidative stress on adenosine receptor expression by nonpigmented ciliary epithelial cells in vitro. Levels of adenosine and its catabolites inosine, hypoxanthine, and xanthine were measured in cell culture supernatants and aqueous humor samples by HPLC. RESULTS: All four adenosine receptor subtypes (A2A > A1 > A2B > A3) were coexpressed but differently distributed in the ciliary epithelium of control eyes, with the A3 receptor being localized to the basolateral membrane infoldings of the nonpigmented epithelial cells. A selective, approximately 10-fold upregulation of A3 receptor mRNA and protein was consistently found in the nonpigmented ciliary epithelium of all PEX eyes, with and without glaucoma, compared with the normal and glaucomatous control eyes. Significant upregulation of A3 receptor message in nonpigmented epithelial cells was induced by both hypoxia and oxidative stress in vitro, together with increased levels of inosine, hypoxanthine, and xanthine in the supernatants. Levels of adenosine and its catabolites, however, were not significantly elevated in the aqueous humor of patients with PEX. CONCLUSIONS: Considering the known role of the A3 adenosine receptor in modulating aqueous humor secretion, its selective, probably hypoxia-induced upregulation in the ciliary epithelium may not only confer cytoprotection but also influence aqueous humor dynamics and may be accessible to therapeutic intervention in patients with PEX.

Myocilin mutations causing glaucoma inhibit the intracellular endoproteolytic cleavage of myocilin between amino acids Arg226 and Ile227.

Myocilin is a secreted glycoprotein of unknown function that is ubiquitously expressed in many human organs, including the eye. Mutations in this protein produce glaucoma, a leading cause of blindness worldwide. To explore the biological role of myocilin and the pathogenesis of glaucoma, we have analyzed the expression of recombinant wild type and four representative pathogenic myocilin mutations (E323K, Q368X, P370L, and D380A) in transiently transfected cell lines derived from ocular and nonocular tissues. We found that wild type myocilin undergoes an intracellular endoproteolytic processing at the C terminus of Arg226. This cleavage predicts the production of two fragments, one of 35 kDa containing the C-terminal olfactomedin-like domain, and another of 20 kDa containing the N-terminal leucine zipper-like domain. Here we have analyzed the 35-kDa processed fragment, and we have found that it is co-secreted with the nonprocessed protein. Western immunoblot analyses showed that human aqueous humor and some ocular tissues also contain the processed 35-kDa myocilin, indicating that the endoproteolytic cleavage occurs in vivo. Mutant myocilins accumulated in the endoplasmic reticulum of transfected cells as insoluble aggregates. Interestingly, the four pathogenic myocilins inhibited the endoproteolytic processing with varying efficiency. Furthermore, the mutation P370L, which produces the most severe glaucoma phenotype, also elicited the most potent endoproteolytic cleavage inhibition. We propose that the endoproteolytic processing might regulate the activity of myocilin and that the inhibition of the processing by pathogenic mutations impairs the normal role of myocilin.

FOXC1 transcriptional regulatory activity is impaired by PBX1 in a filamin A-mediated manner.

FOXC1 mutations underlie Axenfeld-Rieger syndrome, an autosomal dominant disorder that is characterized by a spectrum of ocular and nonocular phenotypes and results in an increased susceptibility to glaucoma. Proteins interacting with FOXC1 were identified in human nonpigmented ciliary epithelial cells. Here we demonstrate that FOXC1 interacts with the actin-binding protein filamin A (FLNA). In A7 melanoma cells possessing elevated levels of nuclear FLNA, FOXC1 is unable to activate transcription and is partitioned to an HP1alpha, heterochromatin-rich region of the nucleus. This inhibition is mediated through an interaction between FOXC1 and the homeodomain protein PBX1a. In addition, we demonstrate that efficient nuclear and subnuclear localization of PBX1 is mediated by FLNA. Together, these data reveal a mechanism by which structural proteins such as FLNA can influence the activity of a developmentally and pathologically important transcription factor such as FOXC1. Given the resemblance of the skeletal phenotypes caused by FOXC1 loss-of-function mutations and FLNA gain-of-function mutations, this inhibitory activity of FLNA on FOXC1 may contribute to the pathogenesis of FLNA-linked skeletal disorders.

Identification of target genes regulated by FOXC1 using nickel agarose-based chromatin enrichment.

PURPOSE: To overcome the problem of antibody availability, often encountered during chromatin immunoprecipitation (ChIP) assays, nickel agarose-based chromatin enrichment (NACE) was developed. Based on the affinity of (His)-6-tagged proteins for the nickel ion, this modified form of ChIP allows the isolation of chromatin in the absence of specific antibodies. METHODS: Nonpigmented ciliary epithelium cells were transfected with (His)-6-tagged FOXC1. FOXC1-enriched chromatin complexes were isolated by using the tight electrostatic interaction between histidine residues of the recombinant FOXC1 protein and nickel. One hundred fifty NACE-enriched clones were sequenced and subjected to in silico and biochemical analyses. RESULTS: Twenty-six clones were detected near known genes: Eight were near predicted but uncharacterized genes, eight were within areas where neither known nor predicted genes have yet been mapped, four were chimeric, and the rest were either repetitive (n=81) or poor-quality (n=23) sequences. Twenty of the 26 known genes were expressed in the eye. Five of the NACE-enriched clones (BMP2K, DACH, FVT-1, SIX-1, and PGE-2 receptor), as well as nine clones selected from the literature, were validated by PCR amplification in two independent lots of NACE-enriched chromatin. All five NACE-selected genes were detected in two independent assays, as well as four (BMP7, SMAD2, TGF-B1, and WNT6) of the nine genes selected from the literature, consistent with these genes' being regulated by FOXC1. CONCLUSIONS: NACE is a useful technique allowing specific chromatin enrichment in cases where antibodies are unavailable. Specific recovery of PTGER, DACH1, WNT6, and FVT-1 implicates FOXC1 in a variety of cellular events including modulation of intraocular pressure, cell cycle, ocular development, and oncogenesis.

Hyposmotic activation of ICl,swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane.

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl(-) conductance (I(Cl,swell)), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the I(Cl,swell) following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl(-) current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as I(Cl,swell). Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of I(Cl,swell). Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to I(Cl,swell) following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.

Sex steroid hormone metabolism takes place in human ocular cells.

Steroids are potentially important mediators in the pathophysiology of ocular diseases. In this study, we report on the gene expression in the human eye of a group of enzymes, the 17beta-hydroxysteroid dehydrogenases (17HSDs), involved in the biosynthesis and inactivation of sex steroid hormones. In the eye, the ciliary epithelium, a neuroendocrine secretory epithelium, co-expresses the highest levels of 17HSD2 and 5 mRNAs, and in lesser level 17HSD7 mRNA. The regulation of gene expression of these enzymes was investigated in vitro in cell lines, ODM-C4 and chronic open glaucoma (GCE), used as cell models of the human ciliary epithelium. The estrogen, 17beta-estradiol (10(-7) M) and androgen agonist, R1881 (10(-8) M) elicited in ODM-C4 and GCE cells over a 24 h time course a robust up-regulation of 17HSD7 mRNA expression. 17HSD2 was up-regulated by estradiol in ODM-C4 cells, but not in GCE cells. Under steady-state conditions, ODM-C4 cells exhibited a predominant 17HSD2 oxidative enzymatic activity. In contrast, 17HSD2 activity was low or absent in GCE cells. Our collective data suggest that cultured human ciliary epithelial cells are able to metabolize estrogen, androgen and progesterone, and that 17HSD2 and 7 in these cells are sex steroid hormone-responsive genes and 17HSD7 is responsible to keep on intra/paracrine estrogenic milieu.

A3 adenosine and CB1 receptors activate a PKC-sensitive Cl- current in human nonpigmented ciliary epithelial cells via a G beta gamma-coupled MAPK signaling pathway.

(1) We examined A3 adenosine and CB1 cannabinoid receptor-coupled signaling pathways regulating Cl(-) current in a human nonpigmented ciliary epithelial (NPCE) cell line. (2) Whole-cell patch-clamp recordings demonstrated that the A3 receptor agonist, IB-MECA, activates an outwardly rectifying Cl(-)current (I(Cl,Aden)) in NPCE cells, which was inhibited by the adenosine receptor antagonist, CGS-15943 or by the protein kinase C (PKC) activator, phorbol 12,13 dibutyrate (PDBu). (3) Treatment of NPCE cells with pertussis-toxin (PTX), or transfection with the COOH-terminus of beta-adrenergic receptor kinase (ct-betaARK), inhibited I(Cl,Aden). The phosphatidyl inositol 3-kinase (PI3K) inhibitor, wortmannin, had no effect on I(Cl,Aden); however, the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, inhibited I(Cl,Aden). (4) Reverse transcription-polymerase chain reaction experiments and immunocytochemistry confirmed mRNA and protein expression for the CB1 receptor in NPCE cells, and the CB1 receptor agonist, Win 55,212-2, activated a PDBu-sensitive Cl(-) current (I(Cl,Win)). (5) Transfection of NPCE cells with the human CB1 (hCB1) receptor, increased I(Cl,Win), consistent with increased receptor expression, and I(Cl,Win) in hCB1 receptor-transfected cells was decreased after application of a CB1 receptor inverse agonist, SR 141716. (6) Constitutive activity for CB1 receptors was not significant in NPCE cells as transfection with hCB1 receptors did not increase basal Cl(-) current, nor was basal current inhibited by SR 141716. (7) I(Cl,Win) was inhibited by PTX preincubation, by transfection with ct-betaARK and by the MEK inhibitor, PD98059, but unaffected by the PI3K inhibitor, wortmannin. (8) We conclude that both A3 and CB1 receptors activate a PKC-sensitive Cl(-) current in human NPCE cells via a G(i/o)/Gbetagamma signaling pathway, in a manner independent of PI3K but involving MAPK.