Cannabidiol protects retinal neurons by preserving glutamine synthetase activity in diabetes.

PURPOSE: We have previously shown that non-psychotropic cannabidiol (CBD) protects retinal neurons in diabetic rats by inhibiting reactive oxygen species and blocking tyrosine nitration. Tyrosine nitration may inhibit glutamine synthetase (GS), causing glutamate accumulation and leading to further neuronal cell death. We propose to test the hypothesis that diabetes-induced glutamate accumulation in the retina is associated with tyrosine nitration of GS and that CBD treatment inhibits this process. METHODS: Sprague Dawley rats were made diabetic by streptozotocin injection and received either vehicle or CBD (10 mg/kg/2 days). After eight weeks, retinal cell death, Müller cell activation, GS tyrosine nitration, and GS activity were determined. RESULTS: Diabetes causes significant increases in retinal oxidative and nitrative stress compared with controls. These effects were associated with Müller cell activation and dysfunction as well as with impaired GS activity and tyrosine nitration of GS. Cannabidiol treatment reversed these effects. Retinal neuronal death was indicated by numerous terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)-labeled cells in diabetic rats compared with untreated controls or CBD-treated rats. CONCLUSIONS: These results suggest that diabetes-induced tyrosine nitration impairs GS activity and that CBD preserves GS activity and retinal neurons by blocking tyrosine nitration.

Endocytosis and degradation of interstitial retinol-binding protein: differential capabilities of cells that border the interphotoreceptor matrix.

Between the pigment epithelium and the outer limiting membrane of the retina is an extracellular compartment filled with the interphotoreceptor matrix (IPM). A prominent component of the IPM is a glycoprotein known as interstitial retinol-binding protein (IRBP). Using in vitro techniques, we compared the ability of the cells that border this compartment to internalize colloidal gold (CG) coated with either IRBP or ovalbumin, a glycoprotein not found in the IPM. Neither IRBP-CG nor ovalbumin-CG was internalized by the Muller's cells. Both rod and cone photoreceptors take up IRBP-CG, which is observed in small vesicles and multivesicular bodies. Neither photoreceptor type takes up ovalbumin-CG. Acid phosphatase cytochemistry indicates that acid phosphatase reaction product in the multivesicular bodies co-localizes with IRBP-CG, which suggests that this molecule is degraded by rod and cone photoreceptors and is not recycled. The pigment epithelium internalizes IRBP-CG and ovalbumin-CG, both of which remain in small cytoplasmic vesicles near the apical plasma membrane. There is no indication that vesicles that contain either IRBP-CG or ovalbumin-CG fuse with the lysosomal system in the pigment epithelial cells during the incubation.

Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter.

We have previously shown that postnatal expression of the viral oncoprotein SV40 T antigen in rod photoreceptors (transgene MOT1), at a time when retinal cells have withdrawn from the mitotic cycle, leads to photoreceptor cell death (Al-Ubaidi et al., 1992. Proc. Natl. Acad. Sci. USA. 89:1194-1198). To study the effect of the specificity of the promoter, we replaced the mouse opsin promoter in MOT1 by a 1.3-kb promoter fragment of the human IRBP gene which is expressed in both rod and cone photoreceptors during embryonic development. The resulting construct, termed HIT1, was injected into mouse embryos and five transgenic mice lines were established. Mice heterozygous for HIT1 exhibited early bilateral retinal and brain tumors with varying degrees of incidence. Histopathological examination of the brain and eyes of three of the families showed typical primitive neuroectodermal tumors. In some of the bilateral retinal tumors, peculiar rosettes were observed, which were different from the Flexner-Wintersteiner rosettes typically associated with human retinoblastomas. The ocular and cerebral tumors, however, contained Homer-Wright rosettes, and showed varying degrees of immunoreactivity to antibodies against the neuronal specific antigens, synaptophysin and Leu7, but not to antibodies against photoreceptor specific proteins. Taken together, the results indicate that the specificity of the promoter used for T antigen and/or the time of onset of transgene expression determines the fate of photoreceptor cells expressing T antigen.

An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis.

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.

Neuroprotective effects of cannabidiol in endotoxin-induced uveitis: critical role of p38 MAPK activation.

PURPOSE: Degenerative retinal diseases are characterized by inflammation and microglial activation. The nonpsychoactive cannabinoid, cannabidiol (CBD), is an anti-inflammatory in models of diabetes and glaucoma. However, the cellular and molecular mechanisms are largely unknown. We tested the hypothesis that retinal inflammation and microglia activation are initiated and sustained by oxidative stress and p38 mitogen-activated protein kinase (MAPK) activation, and that CBD reduces inflammation by blocking these processes. METHODS: Microglial cells were isolated from retinas of newborn rats. Tumor necrosis factor (TNF)-alpha levels were estimated with ELISA. Nitric oxide (NO) was determined with a NO analyzer. Superoxide anion levels were determined by the chemiluminescence of luminol derivative. Reactive oxygen species (ROS) was estimated by measuring the cellular oxidation products of 2', 7'-dichlorofluorescin diacetate. RESULTS: In retinal microglial cells, treatment with lipopolysaccharide (LPS) induced immediate NADPH oxidase-generated ROS. This was followed by p38 MAPK activation and resulted in a time-dependent increase in TNF-alpha production. At a later phase, LPS induced NO, ROS, and p38 MAPK activation that peaked at 2-6 h and was accompanied by morphological change of microglia. Treatment with 1 microM CBD inhibited ROS formation and p38 MAPK activation, NO and TNF-alpha formation, and maintained cell morphology. In addition, LPS-treated rat retinas showed an accumulation of macrophages and activated microglia, significant levels of ROS and nitrotyrosine, activation of p38 MAPK, and neuronal apoptosis. These effects were blocked by treatment with 5 mg/kg CBD. CONCLUSIONS: Retinal inflammation and degeneration in uveitis are caused by oxidative stress. CBD exerts anti-inflammatory and neuroprotective effects by a mechanism that involves blocking oxidative stress and activation of p38 MAPK and microglia.

N-terminal sequence homologies in interstitial retinol-binding proteins from 10 vertebrate species.

We report here the first comprehensive comparative NH2-terminal sequence studies of interstitial retinol-binding protein (IRBPs) from nine mammals (including cattle) and one amphibian. This study has revealed that in many species the N-terminus of IRBP includes a 3-6 amino acid extension. IRBP possessing this leader sequence is sometimes mixed with IRBP from which this sequence has been excised.

Levels of alpha- and gamma-tocopherol in human eyes: evaluation of the possible role of IRBP in intraocular alpha-tocopherol transport.

Alpha-tocopherol was distributed almost equally between the retina and its underlying pigmented layers (pigment epithelium and choroid). Only 8.4% of the total alpha-tocopherol occurred in the iris and ciliary body. Alpha-tocopherol content was expressed as amount per eye, per cm2, and per 100 g wet weight. The combined retina and pigment epithelium-choroid contained 2.9 +/- 1.0 mg/100 g wet weight (means +/- SD, n = 30 donors). Gamma-tocopherol represented 20.9 +/- 12.2 mol % of the alpha-tocopherol. The anterior tissues contained 0.4 +/- 0.2 mg/100 g (n = 19 donors). No significant correlation with age was found. Purified bovine interstitial retinol-binding protein (IRBP) bound exogenous 3H-alpha-tocopherol, which could be displaced by unlabeled all-trans retinol (KD = 10(-6) M). Much higher concentrations of unlabeled alpha-tocopherol were required to achieve a partial displacement of bound 3H-all-trans retinol. No endogenous alpha-tocopherol could be detected in bovine interphotoreceptor matrix.

Precocious IRBP gene expression during mouse development.

PURPOSE: To determine if the time course for the onset of gene and protein expression for interphotoreceptor binding protein (IRBP) precedes that of opsin in the developing mouse retina. METHODS: Relative mRNA levels of the IRBP and opsin genes were determined in prenatal and postnatal retinal RNA with RNase protection analysis (RPA). To determine if IRBP and opsin protein expressions are differentially regulated, dissociated retinal cells from postnatal (P) days 2 and 3 mice, that were injected with BrdU, were then double-labeled with antibodies against BrdU and either opsin or IRBP. RESULTS: With RPA, IRBP mRNA was detected on embryonic (E) day 11 at the time of cone formation, whereas opsin mRNA was not detected until P0. It took until P3 for opsin expression to reach significant levels, whereas rods already appear during embryonic development. IBRP transcription preceded that of opsin because it rapidly increased from E13 to an early postnatal day. By P20, the expression levels of IRBP and opsin achieved constancy. Double antibody labeling revealed positive staining for both IRBP and BrdU as soon as 2 hours after injection, but it took until 40 hours for double positive staining for opsin and BrdU. CONCLUSION: Because only IRBP protein expression was observed before the last mitosis of the photoreceptor precursor cells, IRBP could be essential for retinal development.

Expression of phosphatidylinositol 3-kinase during EGF-stimulated wound repair in rabbit corneal epithelium.

PURPOSE: To investigate the effect of epidermal growth factor (EGF) on the induction of phosphatidylinositol 3-kinase (PI 3- kinase) gene expression during rabbit corneal epithelial wound repair. METHODS: Epithelial wounds (6 mm in size) were created in rabbit corneas and EGF (2 microg) applied every 8 hours to one eye, and the other eye served as a control. The wound repair was monitored by staining the tissue with fluorescein followed by photography. The wound area was quantified with a computer program. At different time intervals, the rabbits were killed and the corneal epithelium used for estimation of PI 3-kinase activity, western blot analysis, or reverse transcription-polymerase chain reaction (RT-PCR). For in situ hybridization, the whole corneas were sectioned and the sections processed with PI 3-kinase mRNA probes. RESULTS: In the untreated eye, the epithelial wound progressively healed in a time-dependent manner, with 75% of the wound closed at 48 hours post wounding. Application of EGF to the corneal epithelium further stimulated wound repair at all time intervals, and the wound was completely closed at 48 hours. Analysis of PI 3-kinase showed a time-dependent increase in its enzyme activity that was maximally increased at 36 hours, the time when the wound was nearly closed. Western blot analysis revealed increased amounts of PI 3- kinase protein during the course of wound repair. Analysis of RT-PCR products from epithelial tissues, taken at different times during wound repair, showed increased PI 3-kinase expression that was maximum at 48 hours post wounding. A visible increase in PI 3-kinase gene expression was also detected by in situ hybridization during the course of the wound repair. This expression was increased maximally by EGF at 48 hours post wounding. CONCLUSIONS: The results indicate a temporal correlation between increased activation and expression of PI 3- kinase and the epithelial wound repair. Topical application of EGF further stimulates the activity and expression of PI 3- kinase. It is suggested that PI 3- kinase and its products may play a role in EGF-induced cell proliferation during corneal epithelial wound repair.

Electron microscopic immunocytochemistry of interstitial retinol-binding protein in vertebrate retinas.

Interstitial retinol binding protein (IRBP) is a soluble glycoprotein found in the interphotoreceptor matrix (IPM) and implicated in shuttling retinol between retina and pigment epithelium (PE) cells. The authors have studied the distribution of IRBP by EM immunocytochemistry. Thin sections of Lowicryl K4M embedded R. pipiens, X. laevis, bovine and human retinas were labeled sequentially with affinity purified rabbit antibovine IRBP, biotinyl-sheep antirabbit F(Ab')2, and avidin-ferritin, or with avidin and biotinyl-ferritin. Antigen was in the interphotoreceptor space and intercalated into the narrow spaces between PE cell microvilli. IRBP penetration between PE cells was delimited abruptly by the PE junctional complexes. IRBP was also observed in small vacuoles in the apical cytoplasm of PE cells and in PE cell phagosomes that contained IRBP surrounding ingested rod tips. IPM was heavily but inhomogeneously labeled. Antigen was usually deposited along the ROS and COS plasma membrane in a confluent layer, but sometimes it was distributed in large (ca. 0.2-micron thick) clumps. In bovine and human retinas, the connecting cilium was ensheathed by antigen at high density but an unlabeled halo surrounded its plasma membrane. The apical plasma membrane of the inner segment aligned along the connecting cilium was also densely coated by antigen. In both frog retinas, the ridges of the periciliary ridge complex (PRC) were coated with antigen. In none of the four species examined was Golgi labeling present. In bovine retinas, labeled vacuoles (granules) in the myoid region were found in very low numbers (15 vacuoles in 358 rod cells). Amphibian retinas also contained only small numbers of myoid vacuoles labeled by anti-IRBP. Absence of antibody binding to intracellular sites of synthesis in any of the cells that abut the interphotoreceptor matrix suggests that the antigen may be masked prior to its release from the synthetic cell(s) or that its level is below limits of detection.

ETB and epidermal growth factor receptor stimulation of wound closure in bovine corneal epithelial cells.

PURPOSE: To determine if there is a heterogeneous pattern of endothelin (ET) receptor subtype (i.e., ETA and ETB) gene expression in the bovine corneal epithelium (BCE). To determine if ET receptor subtype stimulation increases the effectiveness of epidermal growth factor (EGF) to accelerate wound closure in a primary culture of bovine corneal epithelial cells (BCEC). METHODS: In situ hybridization histochemistry was used to characterize ETA and ETB gene expression in the BCE. A wound closure assay evaluated wound healing rates in BCEC after 4 to 7 days in culture. [3H] thymidine incorporation and MTT assay measured proliferation. RESULTS: ETA gene expression was appreciably higher in the basal cells than in the suprabasal cells, whereas the pattern for ETB was reversed. Epidermal growth factor (5 ng/ml) maximally increased wound closure by 145% above the control. With 5 ng/ml EGF, either 10(-9) M ET-1 or 10(-8) M sarafotoxin-6-c (s-6-c) increased wound closure by an additional 39% (P < 0.001) above that measured with 5 ng/ml EGF alone. BQ123 (10(-7) M) did not alter any of these effects of ET-1 or s-6-c. Epidermal growth factor stimulated wound closure through a selective increase in proliferation. Neither ET-1 nor s-6-c alone had any effect on proliferation or migration. CONCLUSIONS: Both ETA and ETB genes are expressed in BCE. However, in BCEC only, ETB stimulation increases the effectiveness of EGF to stimulate wound closure. This response was caused by an increase in cell migration rather than proliferation because, after treatment with mitomycin C, neither ET-1 nor EGF stimulated wound closure.

Endothelin receptor-mediated Ca2+ signaling and isoform expression in bovine corneal epithelial cells.

PURPOSE: Bovine corneal epithelial cells (BCEC) were cultured to determine whether endothelin (ET) receptor subtype stimulation affects ET isoform expression (ET-1, ET-2, and ET-3) through capacitative Ca2+ influx. To probe in the isolated bovine corneal epithelium (BCE) for ET isoform and ET (i.e., ETA and ETB) receptor gene expression. METHODS: [Ca2+]i transients were characterized with microfluorometry. Endothelin isoform and ET receptor gene expression were probed with RNase protection analysis. Enzyme-linked immunosorbent assay was used to measure levels of ET-1-like immunoreactivity (ET-1-LI) in conditioned medium. RESULTS: ET-1 (10(-6) M) increased [Ca2+]i more than twofold. After treatment with 10(-7) M alltrans retinoic acid (an inducer of differentiation), 10(-6) M sarafotoxin (S-6-c) (a selective ETB agonist), had a similar effect. Preincubation with either 5 microM U73122 (an inhibitor of IP8 formation) or 10 microM cyclopiazonic acid, which depletes intracellular Ca2+ store content, eliminated ET agonist-mediated [Ca2+]i increases. With a nominally Ca(2+)-free solution containing 10 microM cyclopiazonic acid, simultaneous 10(-6) M ET-1 and extracellular Ca2+ additions transiently increased [Ca2+]i twofold, whereas 10(-6) M S-6-c increased it by only 20%. This augmentation was eliminated by preexposure to either BQ123 (10 microM), selective ETA receptor antagonist, U73122 (5 microM), or SKF 96365 (3 x 10(-5) M), an inhibitor of stores-operated channels. ET-1, ET-2 isoforms, and ET receptor mRNAs were identified. S-6-c (10(-6) M) increased the level of ET-1-LI after 12 hours by approximately ninefold. CONCLUSIONS: In BCEC, capacitative calcium influx is involved in mediating a positive feedback relationship between ETB receptor stimulation and ET protein expression. Identification of ET-1 and ET-2 gene expression in BCE strengthens the notion that this regulation could be autocrine mediated.

Interstitial retinol-binding protein (IRBP) in the RCS rat: effect of dark-rearing.

The effect of light- and dark-rearing on the amounts of rhodopsin and interstitial retinol-binding protein (IRBP) in RCS rats and their congenic controls (RCS-rdy+) was determined. Rhodopsin was measured spectroscopically and IRBP by dot-blot enzyme immunoassay utilizing rabbit antibovine IRBP IgG. After P15-20, dark-reared RCS and RCS-rdy+ rats always had more rhodopsin than their light-reared, age-matched counterparts. The rhodopsin in the light-reared RCS rats peaked at about 2 nmol/eye at P20-25. The rhodopsin in the dark-reared RCS rats peaked at about 4 nmol/eye at P60-70. Maintenance of RCS-rdy+ rats in darkness had no effect on their IRBP content, which continued to increase up to P80-110. In both groups of RCS rats, the amount of IRBP reached a peak at P22. In RCS rats maintained in darkness, the amount of IRBP attained at this peak was about twice that in the corresponding light-reared group and in RCS-rdy+ animals at this age. The decline of IRBP after P22 in RCS rats was slowed in darkness by approximately 10 days. This slowed decline of IRBP is associated with a decreased rate of photoreceptor degeneration, and the results are therefore consistent with the hypothesis that the photoreceptors synthesize and secrete IRBP. The layer of membranous debris would restrict the diffusion of IRBP in the subretinal space and could partially exclude this retinol transport protein from access to the zone adjacent to the apical surface of the retinal pigment epithelium (RPE).

Rhodopsin, vitamin A, and interstitial retinol-binding protein in the rd chicken.

In order to determine whether blindness in the rd strain of Rhode Island Red chickens is due to a defect in the vitamin A (visual) cycle, spectroscopy, high performance liquid chromatography, and immunochemical techniques were used to compare the amounts of rhodopsin, interstitial retinol-binding protein, and vitamin A compounds in the dark-adapted eyes of homozygous rd and heterozygous carriers. In both groups of chickens, (up to 6 weeks post-hatching) the distribution of stored vitamin A differed from other vertebrates (mammals, amphibians, fish) in that more than half of the retinyl palmitate/stearate occurred in the neurosensory retina. The 11-cis isomer accounted for nearly 100% of the retinyl palmitate/stearate in the neurosensory retinas of both groups. In the pigmented layers (pigment epithelium and choroid) the 11-cis isomer amounted to 70.1 +/- 4.2% in the carrier, and 65.1 +/- 2.9% in the rd birds. With respect to their content of rhodopsin, IRBP, retinyl palmitate/stearate and unesterified retinol, (both 11-cis and all-trans isomers) no significant difference could be demonstrated between the eyes of rd and carrier chickens (3 days and 28 days post-hatching). These results therefore demonstrate that the ocular tissues of rd chickens do not lack IRBP, the putative extracellular transport protein for vitamin A, that these tissues synthesize and store the 11-cis isomer of vitamin A, and that the 11-cis isomer is used to form rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Interstitial retinol-binding protein (IRBP) in subretinal fluid.

Antibodies against bovine interstitial retinol-binding protein (b-IRBP) were used to detect human IRBP (h-IRBP) on immunoblots of eight samples of subretinal fluid (SRF) from patients with retinal detachments of between 2 days' and more than 2 years' duration. Using this sensitive technique, it was found that seven of the samples contained h-IRBP in concentrations estimated to range from below 5% up to 19% of normal human IPM. One of these samples displayed two immunoreactive bands of roughly equal intensity, one at a molecular weight of 135,000 (h-IRBP), the other at 115,000. The latter may have been generated by proteolytic cleavage. No h-IRBP could be detected in an eighth sample from a patient with retrolental fibroplasia. It is concluded that the reduced concentration of h-IRBP in SRF may be due to a number of factors that include dilution, proteolytic degradation, and metabolic inactivation of photoreceptors at the detachment site.

Regulation of interphotoreceptor retinoid-binding protein (IRBP) gene expression by cAMP in differentiated retinoblastoma cells.

PURPOSE: To determine the mechanism of cyclic AMP (cAMP) regulation of the interphotoreceptor retinoid-binding protein (IRBP) gene in retinoblastoma cells. METHODS: WERI-Rb1 cells pretreated with laminin or grown on poly-D-lysine-coated substratum for three days were treated with forskolin/3-isobutyl-1-methylxanthin (IBMX) or with dimethyl sulfoxide (DMSO). During a time course of 96 h, cell morphologies were determined by light microscopy, cellular cAMP levels measured by radioimmunoassay, and IRBP and b-actin gene expression determined by Northern blot and RNase protection analyses. IRBP expression and b-actin gene expression in these cells were also determined in the presence or absence of actinomycin D or cycloheximide. RESULTS: After laminin treatment for 3 days, 27-34% of WERI-Rb1 cells differentiated into spindle shapes. Further forskolin treatment in the presence of IBMX for 5 days resulted in many cells exhibiting the formation of long, ramifying, neurite-like processes that were abolished by an inhibitor of protein kinase A. Cells grown on poly-D-lysine-coated substratum treated with forskolin remained undifferentiated. Treatment of laminin-pretreated cells with forskolin/IBMX, but not with DMSO/IBMX, raised the cAMP level 15-fold within the first hour of treatment. Northern blot analysis of these cells showed a rapid increase of IRBP mRNA, but not b-actin mRNA, reaching a maximum of about 3-fold at 6-8 h. A similar increase of IRBP mRNA was observed using RNase protection analysis except that the maximum was observed at 2-4 h. Actinomycin D blocked this IRBP mRNA induction. Cycloheximide had no effect in this induction. CONCLUSIONS: These results demonstrate that laminin induces WERI-Rb1 cell differentiation and cAMP provokes the formation of long ramifying neurite-like processes. Forskolin selectively induces IRBP gene expression in the laminin-treated cells through a cAMP-mediated pathway without de novo protein synthesis.

Ultrastructural and ERG findings in mice with adenomatous polyposis coli gene disruption.

PURPOSE: In order to continue the previous morphological studies of eyes from mice with adenomatous polyposis coli (APC) gene mutation at codon 1638, we determined the ultrastructural and electrophysiologic characteristics of these eyes. METHODS: Thirty-eight eyes from 20 mice heterozygous for APC gene mutation and 22 eyes from 11 wild-type mice were examined by light microscopy. Six APC-modified eyes without light microscopic abnormalities, four APC-modified eyes with focal light microscopic abnormalities, and four wild-type eyes were examined by electron microscopy. Electroretinograms were recorded from four APC-modified and three wild-type mice. RESULTS: Four of 38 APC-modified eyes demonstrated ultrastructural evidence of focal RPE cells with increased melanosome production and atrophy. Other areas of the RPE in these four eyes demonstrated no ultrastructural abnormalities. Three APC-modified eyes demonstrated electron and light microscopic evidence of RPE hyperplasia. Electron microscopic examination of APC-modified eyes without light microscopic evidence of abnormalities demonstrated no ultrastructural differences from age-matched controls. Electroretinography demonstrated no differences in the b-wave or c-wave amplitudes between APC-modified and wild-type mice. CONCLUSIONS: While light microscopic RPE alterations are observed in these APC-modified mice, the absence of a generalized, ultrastructural murine RPE defect is in contradistinction to observations in electron microscopic investigations of humans with colonic polyposis, pigmented ocular fundus lesions, and APC gene mutations between codons 463 and 1444. Our results in mice with APC mutation at codon 1638, however, are consistent with a previously identified association between the expression of pigmented ocular fundus lesions and region-specific mutation in the human APC gene. The APC protein may possess a physiologic function for both retinal and RPE development.

Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone-rod-homeobox element is essential for photoreceptor-specific expression in vivo.

The essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and +19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a "cone-rod-homeobox element" (CRXE) and a "photoreceptor conserved element" (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.

Interstitial retinol-binding protein (IRBP) in retinoblastoma.

Interstitial retinol-binding protein (IRBP) is an extracellular glycoprotein that appears to be synthesized by the photoreceptors in the normal retina and may be involved in shuttling retinoids through the interphotoreceptor matrix between the retina and pigment epithelium. The present work demonstrated immunochemically that IRBP of the same molecular weight as normal human IRBP (135,000) was present in three retinoblastomas, irrespective of their degree of differentiation. Tumor 1 was classified as differentiated; Tumors 2 and 3 were classified as poorly differentiated. The level of IRBP in the soluble proteins of Tumor 2 was about 8 times that in Tumor 1 and about one-half that in the soluble proteins (including adhering interphotoreceptor matrix) from a pair of normal retinas from a 31-year-old donor. IRBP occurred in the interstitial space of Tumor 3. Most of the IRBP in this tumor was recovered from the medium in which the undifferentiated cells were dispersed. Incubation of the isolated cells from Tumor 3 in medium containing [(3)H]leucine demonstrated that [(3)H]IRBP was secreted into the medium. The [(3)H]IRBP was immunoreactive with rabbit antibovine IRBP antibodies. It was inferred that the [(3)H]IRBP was glycosylated because it was bound by immobilized concanavalin A and could be displaced with ?-methylmannopyranoside. Since IRBP is not normally found in retinas until the time of photoreceptor differentiation, regulation of its gene may be defective in this malignant neuroectodermal neoplasm. The present findings are relevant to the possible role of retinoids and their binding proteins in neoplastic cells, because they demonstrate for the first time the presence of an extracellular retinoid-binding protein in tumor tissue.

Retinoid-binding proteins and retinol esterification in cultured retinal pigment epithelium cells.

The esterification of all-trans retinol and the occurrence of cytosolic retinoid-binding proteins was investigated in cultured bovine retinal pigment epithelium (RPE) cells. (3)H-labeled all-trans retinyl ester (mainly palmitate) was formed at an initial rate of 0.1 nmol.mg protein(?1).min(?1) when (3)H-labeled all-trans retinol was incubated with the 100,000 g pellet obtained from a homogenate of freshly-harvested cells. No esterification could be detected under the same conditions after 14 days in culture in defined medium (DM) or in medium containing 20% fetal bovine serum (CM). No enhancement or restoration of esterifying capacity was observed when the assay mixture was supplemented with palmitoyl CoA. As determined by specific, saturable binding of (3)H-labeled all-trans retinol and (3)H-labeled 11-cis retinal to proteins with mol. wts 16,000 and 33,000 dalton on calibrated Bio-Sil TSK 250 size-exclusion columns, the cytosol of freshly-harvested RPE cells contained cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRAlBP). By comparison with the quantity of (3)H-labeled all-trans retinol bound under identical conditions to pure dog liver CRBP, it was estimated that fresh RPE cells contained 102 +/- 3 ng CRBP.?g cytosol protein(?1). In cultured and subcultured cells, CRBP was present at much lower levels (down to one-tenth of the initial amounts) and CRAlBP could not be detected. Since binding of (3)H-labeled all-trans retinoic acid to a protein with molecular weight of 17,000 dalton was not observed in the cytosols of fresh or cultured cells, it was concluded that cellular retinoic acid binding protein (CRABP) was either present at very low levels or absent altogether. An unidentified peak of specific (3)H-labeled all-trans-retinoic acid binding at mol. wt 61,000 dalton was prominent in subcultured cells. These results show that in RPE cells in culture the expression of differentiated phenotype with respect to retinoid utilization undergoes significant modification. It is postulated that changes in the composition of the extracellular matrix (e.g. absence of interstitial retinol-binding protein, IRBP) may be involved.