Miguel Coca-Prados

Sequence Analysis and Homology Modeling Suggest That Primary Congenital Glaucoma on 2p21 Results from Mutations Disrupting Either the Hinge Region or the Conserved Core Structures of Cytochrome P4501B1

We recently reported three truncating mutations of the cytochrome P4501B1 gene (CYP1B1) in five families with primary congenital glaucoma (PCG) linked to the GLC3A locus on chromosome 2p21. This could be the first direct evidence supporting the hypothesis that members of the cytochrome P450 superfamily may control the processes of growth and differentiation. We present a comprehensive sequence analysis of the translated regions of the CYP1B1 gene in 22 PCG families and 100 randomly selected normal individuals. Sixteen mutations and six polymorphisms were identified, illustrating an extensive allelic heterogeneity. The positions affected by these changes were evaluated by building a three-dimensional homology model of the conserved C-terminal half of CYP1B1. These mutations may interfere with heme incorporation, by affecting the hinge region and/or the conserved core structures (CCS) that determine the proper folding and heme-binding ability of P450 molecules. In contrast, all polymorphic sites were poorly conserved and located outside the CCS. Northern hybridization analysis showed strong expression of CYP1B1 in the anterior uveal tract, which is involved in secretion of the aqueous humor and in regulation of outflow facility, processes that could contribute to the elevated intraocular pressure characteristic of PCG.

Crocin, safranal and picrocrocin from saffron (Crocus sativus L.) inhibit the growth of human cancer cells in vitro

Extracts of saffron (Crocus sativus L.) have been reported to inhibit cell growth of human tumor cells. In order to study the cytotoxic effect of the characteristic compounds of saffron spice, we have isolated crocin, crocetin, picrocrocin and safranal. Doses inducing 50% cell growth inhibition (LD50) on HeLa cells were 2.3 mg/ml for an ethanolic extract of saffron dry stigmas, 3 mM for crocin, 0.8 mM for safranal and 3 mM for picrocrocin. Crocetin did not show cytotoxic effect. Cells treated with crocin exhibited wide cytoplasmic vacuole-like areas, reduced cytoplasm, cell shrinkage and pyknotic nuclei, suggesting apoptosis induction. Considering its water-solubility and high inhibitory growth effect, crocin is the more promising saffron compound to be assayed as a cancer therapeutic agent.

Cloning and characterization of subtracted cDNAs from a human ciliary body library encoding TIGR, a protein involved in juvenile open angle glaucoma with homology to myosin and olfactomedin

A group of cDNAs isolated from a subtractive ciliary body library of a normal human eye donor revealed 100% identity with TIGR a candidate gene responsible for juvenile open angle glaucoma [Science 275 (1997) 668–670]. Several structural features of the deduced human protein have been noted: a cleavable N‐terminal signal peptide, a periodic repetition at the N‐terminus of leucine and arginine residues at every seventh and eleven position respectively in helix conformation (leucine zipper‐like motif) exhibiting homology with myosin, and with olfactomedin in the C‐terminus. The mRNA for TIGR is abundantly expressed in the ciliary body, iris, heart and skeletal muscle.

Production, purification, and functional analysis of recombinant human and mouse 17β-hydroxysteroid dehydrogenase type 7 ☆

17beta-Hydroxysteroid dehydrogenases (17HSDs) have a central role in the regulation of the biological activity of sex steroid hormones. There is increasing evidence that in addition to their importance in gonads, these hormones also have substantial metabolic roles in a variety of peripheral tissues. In the present study, the cDNA of human 17HSD type 7 was cloned. In silico, the gene corresponding to the cDNA was localized on chromosome 1q23, close to the locus of hereditary prostate cancer 1 (HPC1) (1q24-25) and primary open-angle glaucoma (GLC1A) (1q23-25). Further, a pseudogene was found on chromosome 1q44, close to the locus of predisposing for early-onset prostate cancer (PCaP) (1q42.2-43). Both human (h17HSD7) and mouse 17HSD type 7 (m17HSD7) were for the first time produced as recombinant proteins and purified for functional analyses. Further, kinetic parameters and specific activities were described. h17HSD7 converted estrone (E1) to a more potent estrogen, estradiol (E2), and dihydrotestosterone (DHT), a potent androgen, to an estrogenic metabolite 5alpha-androstane-3beta, 17beta-diol (3betaA-diol) equally, thereby catalyzing the reduction of the keto group in either 17- or 3-position of the substrate. Minor 3betaHSD-like activity towards progesterone (P) and 20-hydroxyprogesterone (20-OH-P), leading to the inactivation of P by h17HSD7, was also detected. m17HSD7 efficiently catalyzed the reaction from E1 to E2 and moderately converted DHT to an inactive metabolite 5alpha-androstane-3alpha,17beta-diol (3alphaA-diol) and to an even lesser degree 3betaA-diol. The mouse enzyme did not metabolize P or 20-OH-P. The expression of 17HSD type 7 was observed widely in human tissues, most distinctly in adrenal gland, liver, lung, and thymus. Based on the enzymatic characteristics and tissue distribution, we conclude that h17HSD7 might be an intracrine regulator of steroid metabolism, fortifying the estrogenic milieu in peripheral tissues.

Association of ClC-3 channel with Cl- transport by human nonpigmented ciliary epithelial cells.

Abstract. Electrophysiologic and volumetric evidence link the swelling-activated Cl− channels [gCl(Vol)] of nonpigmented ciliary epithelial (NPE) cells with the Cl−- channel/Cl−-channel regulator protein pICln. However, inhibitors (verapamil and dideoxyforskolin) of another Cl− channel/regulator (MDR1) have been found to inhibit the volume-activated transport response [the regulatory volume decrease (RVD)] of bovine NPE cells. We have addressed the possible molecular basis for the NPE Cl− channels by volumetric measurements of ODM human NPE cells in hypotonic and isotonic test solutions, and by polymerase chain reaction (PCR) cloning and Northern analyses of the same cells. Verapamil and dideoxyforskolin did inhibit the RVD. However, at a concentration (100 μm) which blocks >90% of the MDR1-associated Cl− currents, forskolin had no effect on the volume-activated Cl− channels or on the inhibition of those channels by protein kinase C. High concentrations of ATP (3.5 and 10 mm) and niflumic acid (IC50∼ 200 μm) also block [gCl(Vol)]. The RVD is inhibited by 9-phenylanthranilic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), unaffected by anthracene-9-carboxylic acid (9-AC), and stimulated by ionomycin. The Cl−-channel blockers NPPB, niflumic acid, DPC and 9-AC, and the Ca2+-ionophore ionomycin had qualitatively similar effects on the rate of staurosporine-activated isotonic cell shrinkage. These results support the concept that the volume-sensitive protein pICln regulates the Cl− channels, and that the same conduits subserve volume- and staurosporine-activated Cl− release. Of the cloned and sequenced Cl− channels, ClC-3 uniquely conforms to the stationary currents and PKC sensitivity of the NPE Cl− channels. PCR amplifications of human cDNA libraries from ciliary body, NPE cells and retina with primers based on human ClC-3 and ClC-4 cDNA, and Northern analyses using the products generated indicated that ciliary epithelial cells express transcripts for ClC-3 (but not ClC-4). We suggest that ClC-3 provides the same conduit for both volume-activated and isotonically staurosporine-activated Cl− channels of human nonpigmented ciliary epithelial cells.

Differential gene expression in the human ciliary epithelium

The generation of expression and subtractive libraries from the ocular ciliary body and cultured ciliary epithelial cells has been instrumental in the cloning, identification and characterization of many genes which, overall reflect a representative profile of transcripts expressed in ciliary nonpigmented, ciliary pigmented and ciliary muscle cells. The cell-specific expression of some of these genes (i.e. a neurotrophic factor, a gene associated with juvenile open glaucoma, and a visual component) reveal a degree of cell differentiation with a diversity of functions and properties higher than previously thought. The protection from light-induced oxidative reactions, free radicals and detoxification, may be partially attributed to the high level of expression in the ciliary epithelium of antioxidative enzymes (i.e., glutathione S-transferase, glutathione peroxidases, selenoprotein-P). The expression of genes encoding plasma proteins (i.e., complement component C4, alpha2-macroglobulin, apolipoprotein D) is in contrast with the view that plasma proteins in aqueous humor are synthesized outside the eye (i.e., liver). The identification of neuropeptide-processing enzymes (i.e., prohormone convertases, carboxypeptidase E, peptidyl-glycine-alpha-amidating monoxigenase), neuropeptides (i.e., secretogranin II, neurotensin) and regulatory peptides (i.e., atrial natriuretic peptide and angiotensinogen) with hypertensive and hypotensive activities provide the molecular basis to support the view that the ciliary epithelium is a neuroepithelium with neuroendocrine functions. We propose a working model to demonstrate that aqueous humor and intraocular pressure are under neuroendocrine control through regulatory peptides synthesized and released by the ciliary epithelium and targeting the peptide producing cells at the inflow system by an autocrine mechanism and/or cells at the outflow system (i.e., trabecular meshwork cells) by a paracrine mechanism. Finally, we hypothesize that these mechanisms could be entrained in the light-dark cycle following the circadian rhythm of aqueous humor and intraocular pressure.

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

ABSTRACT 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 HP1α, 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.

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-μ M 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.

Mammalian ciliary-body epithelial cells in culture produce transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) has recently been found in the aqueous humor. The present study was initiated to test whether the ciliary-body epithelium, the site of aqueous humor formation, is capable of producing TGF-beta. Human, rat and bovine ciliary epithelial cells were grown in tissue culture and their supernatants were tested for TGF-beta using a bioassay. After acid activation of the samples, TGF-beta activity was found in the supernatants of all three mammalian ciliary epithelial cells. Most of this activity could be blocked by a neutralizing antibody against TGF-beta type 2. Native supernatants did not contain detectable TGF-beta activity. Thus, the ciliary epithelium is capable of producing the inactive form of TGF-beta (mainly type 2) and may be a source of TGF-beta in the aqueous humor.