From the Adirondack Biomedical Research Institute,
Lake Placid, New York 12946, the ** Department of Anatomy and Cell
Biology, University of Texas Southwestern Medical Center, Dallas, Texas
75235, and the ¶ Department of Ophthalmology, Wilmer Eye
Institute, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21287
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ABSTRACT |
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Cellular retinaldehyde-binding protein (CRALBP)
is abundantly expressed in the retinal pigment epithelium (RPE) and
Müller cells of the retina, where it is thought to function in
retinoid metabolism and visual pigment regeneration. Mutations in human CRALBP that destroy retinoid binding have been linked to autosomal recessive retinitis pigmentosa. To identify the DNA elements that regulate expression of the human CRALBP gene in the RPE, transient transfection studies were carried out with three CRALBP-expressing human RPE cell culture systems. The regions from 2089 to
1539 base
pairs and from
243 to +80 base pairs demonstrated positive regulatory
activity. Similar activity was not observed with cultured human breast,
liver, or skin cells. Since sequence analysis of the
243 to +80
region identified the presence of two photoreceptor consensus element-1
(PCE-1) sites, elements that have been implicated in photoreceptor gene
regulation, the role of these sequences in RPE expression was examined.
Mutation of either PCE-1 site significantly reduced reporter activity,
and mutation or deletion of both sites dramatically reduced activity.
Electrophoretic mobility shift analysis with RPE nuclear extracts
revealed two complexes that required intact PCE-1 sites. These studies
also identified two identical sequences (GCAGGA) flanking PCE-1, termed
the binding CRALBP element (BCE), that are also important for complex
formation. Southwestern analysis with PCE-1/BCEcontaining probes
identified species with apparent masses near 90-100 and 31 kDa. These
results begin to identify the regulatory regions required for RPE
expression of CRALBP and suggest that PCE-1-binding factor(s) may play
a role in regulating RPE as well as photoreceptor gene expression.
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INTRODUCTION |
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The retinal pigment epithelium (RPE)1 is a polarized monolayer of cells between the photoreceptors and choroid of the eye. Its primary function is maintaining and nourishing the rod and cone photoreceptor cells, including the synthesis of 11-cis-retinal for phototransduction (1). The critical importance of the RPE to the health of photoreceptors has been further illustrated by recent findings that mutations in either of two genes differentially and abundantly expressed in the RPE, namely the cellular retinaldehyde-binding protein (CRALBP) and RPE-65, can cause early onset autosomal recessive retinitis pigmentosa (2-4). The in vivo functions of CRALBP and RPE-65 have yet to be clarified; however, they are both thought to be involved in vitamin A metabolism. A retinal degeneration possibly associated with gene regulation in the RPE has been described for the vitiligo mouse; the vitiligo mouse exhibits genetic defects in transcription factor Mi and may also suffer from abnormal vitamin A metabolism (5).
CRALBP appears to serve as a substrate carrier protein in the mammalian visual cycle, modulating whether 11-cis-retinol is stored as an ester in the RPE or oxidized by 11-cis-retinol dehydrogenase to 11-cis-retinal for visual pigment regeneration (6, 7). The protein carries 11-cis-retinal and/or 11-cis-retinol as an endogenous ligand in the RPE and Müller cells of the retina (7). Notably, the R150Q CRALBP mutation linked to autosomal recessive retinitis pigmentosa destroys the retinoid binding functionality of the protein (2). CRALBP is also present in ciliary body, cornea, and transiently in iris, but not in rod and cone photoreceptors. Within the ciliary epithelium, an anterior to posterior gradient of CRALBP expression exists with more abundant expression in the pars plicata region relative to the pars plana region (8). Recently, CRALBP has been found in oligodendrocytes of the optic nerve and brain, but without retinoid ligands, and CRALBP is also expressed in glia cells of the pineal gland (9). During development, CRALBP is expressed in the rat RPE at E13 and in Müller cells at P1 (10), before 11-cis-retinoids or the isomerase responsible for generating 11-cis-retinoids can be found in the RPE (9). Apparently, the protein serves functions unrelated to visual pigment regeneration in brain and tissues not involved in the visual cycle and may bind ligands other than retinoids.
Due to the apparent importance of CRALBP in the visual cycle, its role
in retinal disease, and its usefulness as a model system for the study
of RPE gene regulation, we have begun to explore the mechanisms
controlling its expression in the RPE. The structure of
RLBP1, the gene encoding human CRALBP, has been reported,
including the transcription start site and 3130 bases of sequence from
the 5'-flanking region (11). It is composed of eight exons, with untranslated regions in exons 1, 2, and 8, and is located on human chromosome 15q26 (12). Previously, we noted the existence of two
photoreceptor consensus elements between CRALBP gene positions 170
and
141
(5'-GAAGGCACTTAGGCAGGACATTTAGGCAGG-3') (11).
The consensus sequence CAATTAG, designated PCE-1 in vertebrates and RCS-1 in Drosophila, has been identified in a number of
mammalian retina-specific genes (e.g. arrestin,
interphotoreceptor retinoid-binding protein, rhodopsin, and red and
green opsin) and in Drosophila rhodopsin genes and has been
proposed to direct photoreceptor cell-specific expression (13).
Mutation of PCE-1 sites in gene constructs from the proximal promoters
of arrestin (14) and interphotoreceptor retinoid-binding protein (15)
also appears to significantly reduce photoreceptor reporter gene
expression in transgenic mice. The PCE-1-rich region of the
RLBP1 proximal promoter exhibits significant homology to the
26-bp Ret-1 element in the rat opsin gene, which has been reported to
direct opsin expression in rod photoreceptors in vivo (16).
In this study, efforts have been initiated to determine the mechanisms
controlling CRALBP cell-specific expression. The possibility was
considered that PCE-1 might also regulate gene expression in the RPE,
not just in photoreceptor cells. We report here transient transfection results, nuclear protein binding studies, and Southwestern analyses that suggest that PCE-1 influences CRALBP gene expression in the RPE.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- Early passage nontransformed human RPE cell cultures (17) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and 10% fetal bovine serum (Intergen Co.) and, for some transfections, were grown on plates coated with Matrigel (Upstate Biotechnology, Inc.) or bovine corneal endothelial cell matrix (17). The spontaneously immortalized human D407 RPE cell line (18) was grown in Dulbecco's modified Eagle's medium and 2% fetal bovine serum plus 210 mM Me2SO (Fluka). The nontransformed human ARPE-19 RPE cell line (19) was obtained as a gift from Dr. L. Hjelmeland (University of California, Davis, CA) and grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Human breast (MCF-7), human liver (HepG2), and human skin fibroblast transformed cell lines (available through the American Type Culture Collection) were grown in minimum Eagle's medium (Life Technologies, Inc.) and 10% fetal bovine serum.
Western Analysis-- Human retinal tissue was obtained from Dr. Arthur Polans and the RS Dow Neurological Sciences Institute/Good Samaritan Hospital (Portland, OR). Extracts from RPE cell cultures and human retinas were prepared with Laemmli SDS-PAGE sample buffer (40) in the presence of protease inhibitors (25 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine), boiled for 4 min, electrophoresed on 12% gels according to Laemmli (40), and electroblotted onto nitrocellulose membrane (Schleicher & Schuell). Membranes were probed with IgG-purified polyclonal anti-CRALBP peptide antibodies S17L, K25Q, and S31F as described previously (20), except that immunoreactivity was detected with the commercial enhanced chemiluminescence system (Amersham Corp.). Human recombinant CRALBP (Mr = 36,343), used as a control, was produced in bacteria using the pET3a vector as described (21). The protein concentration of cell extracts was measured using the BCA protein assay (Pierce).
Reverse Transcription-PCR-- RNA was isolated from RPE cell cultures using the RNAzol protocol (Cinna-Biotec), and first strand synthesis was performed using reverse transcriptase and the Superscript preamplification system (Life Technologies, Inc.). PCR of resulting cDNA templates was performed with a hot start at 94 °C for 1 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 45 s, and 72 °C for 1 min. The primers used were CCCTGACTTCCTATCCTAGG and CTTCTGCAAGGTGTGG, designed upon human CRALBP gene positions 823-842 and 1318-1333, respectively, and span intron 2 (gene position 935-1204). PCR products were evaluated by Southern analysis using a 241-bp 32P-labeled probe constructed by PCR using the human CRALBP cDNA as a template and the above primers.
CRALBP-Reporter Gene Constructs--
Promoter regions of
RLBP1 were amplified by PCR from a 4.7-kb 5'-genomic
subclone (11), and the resulting products were ligated upstream of the
luciferase reporter gene in the pGL2 vector (Promega). Seven wild-type
CRALBP-luciferase reporter gene constructs were created based on size
and orientation; wild-type constructs were designated 0.2, 0.3, 0.3R,
0.8, 1.6, 2.1, and 2.3, where R indicates reverse orientation and the
number reflects the approximate size of the CRALBP promoter region in
kilobases. The 3'-end of all the constructs was RLBP1
position +80; the 5'-end varied as indicated under "Results." Eight
mutant CRALBP-luciferase reporter gene constructs were also created and
are defined under "Results." Mutant constructs containing 4-5
altered nucleotides in individual PCE sites were created with
single-stranded DNA according to Kunkel et al. (22) and
designated pce-dM (for mutation in the distal PCE at RLBP1
positions 165 to
159) and pce-pM (for mutation in the proximal PCE
at RLBP1 positions
152 to
146). Briefly, phosphorylated
mutant oligonucleotides were annealed to single-stranded DNA prepared
in Escherichia coli dut
,
ung
CJ236 cells from the 0.3-kb wild-type reporter
gene construct. The oligonucleotide primers and corresponding
RLBP1 gene positions used to prepare pce-dM and
pce-pM were
183GTGAAGGGTTCTTGAAGGCtgcagGGCAGGACATTTAGGCAG
142
and
167GGCACTTAGGCAGGAtcTgcAGGCAGGAGAGAAAACCTGG
128,
respectively. These primers introduced a PstI
restriction site in place of PCE-1, which facilitated screening of
mutants and generation of pce-2M and pce-
M. Second strand synthesis
was performed with T4 polymerase, and the mutant plasmid was amplified
in E. coli dut+, ung+
DH5
cells. To create mutant constructs with altered bases in both
PCE sites, complementary oligonucleotides containing the mutations were
synthesized (69 and 61 nucleotides, respectively), annealed, and
ligated into the 0.3pce-pM mutant construct following digestion with
NcoI/PstI, yielding mutant construct 0.3pce-2M. To create a deletion mutant lacking both PCE sites, the 93-bp fragment
excised from 0.3pce-pM by PstI digestion was ligated into
0.3pce-dM following PstI digestion, yielding mutant
construct 0.3pce-
M. NcoI/HindIII fragments
from the 0.3-kb series of mutants were ligated into the 2.1-kb
wild-type reporter gene construct following
NcoI/HindIII cleavage, yielding the corresponding
2.1-kb series of mutant constructs. The structures of all reporter
constructs were verified by automated fluorescent DNA sequence
analysis.
Transient Transfections-- Cells were transfected 12-24 h after plating in 6-well plates using either the LipofectAMINE/Lipofectin protocol (Life Technologies, Inc.) or the calcium phosphate procedure (23). Cell extracts and medium were assayed for reporter activity ~48 h after transfection. Transfections of cells utilized 1.75 µg of reporter construct and 0.25 µg of pCMV-SEAP or pXGH5 control construct (Nichols Diagnostics, Inc.) to normalize transfection efficiency to secreted alkaline phosphatase or human growth hormone expression, respectively. Luciferase luminescence was quantified in cell extracts with an EC & G Berthold LB 96P luminometer in the presence of 210 mM coenzyme A. Protein concentration in cell extracts was determined using the BCA assay. For data interpretation, luciferase activity (relative luciferase units/µg of protein cell extract) was normalized to the internal control (secreted alkaline phosphatase or human growth hormone) to correct for variation in transfection efficiency, and results from separate experiments were averaged following normalization to the luciferase activity exhibited by the 2.1-kb wild-type construct.
Preparation of Nuclear Extracts-- Nuclear extracts for EMSA were prepared essentially according to Andrews and Faller (24). Bovine retinal tissue was obtained from JA & WL Lawson Co. (Lincoln NE); rat seminal vesicle extract was obtained from Dr. Martin Tenniswood (Adirondack Biomedical Research Institute, Lake Placid, NY). Briefly, tissue (1 g) was homogenized in 2 ml of 50 mM Tris-HCl (pH 7.5), 25 mM KCl, 5 mM MgCl2, 200 mM sucrose, 0.2 mM phenylmethylsulfonyl fluoride, and 0.2 mM benzamidine. Following bench-top centrifugation (500 × g for 10 min at 4 °C), the pellet fraction was resuspended in 400 µl of cold 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, and 0.25 mM each phenylmethylsulfonyl fluoride and benzamidine. For cell cultures, confluent cells were scraped into 1.5 ml of cold phosphate-buffered saline, centrifuged at 13,000 × g for 2 min, and resuspended as described above. After a 10-min incubation on ice for lysis, cells were vortexed for 10 s, and nuclei were pelleted by centrifugation (13,000 × g). For high salt extraction of nuclear proteins, pellets were incubated for 20 min at 4 °C in 100 µl of 20 mM HEPES-KOH (pH 7), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.25 mM each phenylmethylsulfonyl fluoride and benzamidine and then centrifuged, and the supernatant fraction was harvested. Protein concentration in cell extracts was determined using the BCA assay.
EMSAs-- Oligonucleotide probes (see Table I) were synthesized with 5'-overhangs and radioactively labeled by Klenow fill-in reactions (Promega). Nuclear extracts were incubated with a 2000-fold molar excess of poly(dI-dC) (Pharmacia Biotech Inc.) at room temperature for 10-30 min in 100 mM Tris-HCl (pH 7.4), 50 mM dithiothreitol, 50 mM EDTA, and 22% glycerol (plus unlabeled probe in competition assays) before adding 1-10 ng of labeled probe. After an additional 10-30-min incubation at room temperature, the reactions were stopped and electrophoresed on 4-6% nondenaturing polyacrylamide gels, followed by autoradiography.
Southwestern Analysis--
Southwestern analysis was performed
according to published methods (25, 26) with modifications. Nuclear
extract from human D407 RPE cells and prestained molecular mass
standards (Bio-Rad) were fractionated by SDS-PAGE according to Laemmli
(40) on 8% gels under reducing conditions and electroblotted onto
nitrocellulose membrane (overnight at 4 °C using 50 mA of constant
voltage), and the membrane was stained briefly with Ponceau S. Membrane-immobilized nuclear proteins were denatured in Southwestern
(SW) buffer (25 mM HEPES (pH 7.9), 50 mM KCl, 3 mM MgCl2, and 5 mM dithiothreitol) containing 6 M guanidine hydrochloride for 30 min at
4 °C and then slowly renatured. The renaturation process involved
successive dilutions and incubations (2 × 15 min, 4 °C) in SW
buffer, reducing the guanidine hydrochloride molarity to 3, 1.5, 0.75, 0.38, and 0.18 and finally to zero. Following renaturation, the
membranes was incubated for 4-6 h at 4 °C in SW buffer containing
5% nonfat dried milk. For binding studies, replicate lanes of the blot
were cut into vertical strips and incubated overnight at 4 °C in SW buffer containing 4 µg of poly(dI-dC) and radiolabeled DNA probe; unlabeled DNA probes were included for competition studies. The strips
were then washed with three changes of SW buffer, and the membrane was
reassembled and subjected to autoradiography at 80 °C overnight
with an intensifying screen. Molecular mass markers (Bio-Rad) included
lysozyme (18.5 kDa), soybean trypsin inhibitor (27.5 kDa), carbonic
anhydrase (32 kDa), ovalbumin (49.5 kDa), bovine serum albumin (80 kDa), and phosphorylase b (106 kDa).
Oligonucleotide Synthesis and DNA Sequence Analysis-- Oligonucleotide synthesis and DNA sequence analysis were performed in the Molecular Biology Core Facility of the Adirondack Biomedical Research Institute using Perkin-Elmer, Applied Biosystems Division instrumentation and reagents (Model 393 DNA/RNA synthesizer, Model 373 DNA Sequencer, and the PRISM Dye Terminator Cycle Sequencing Ready Reaction kit). Interpretation of DNA sequence results was facilitated by the use of Sequencher 3.0 software (Gene Codes). Human CRALBP gene sequence upstream of the translation start site was inspected for consensus transcription factor-binding sites manually and by computer using SIGNAL SCAN 2 and the Ghosh transcription factor data base (27).
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RESULTS |
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Expression of CRALBP in Cultured RPE Cells-- Three human RPE cell culture systems were used to study regulation of RLBP1, namely early passage nontransformed RPE cell cultures (17) and RPE cell lines ARPE-19 (19) and D407 (18). Previous reports have shown by Western and Northern analyses that the early passage nontransformed RPE cultures and ARPE-19 cells express CRALBP (17, 19). We used Western analysis and reverse transcription-PCR to confirm the CRALBP expression in D407 RPE cells previously detected by immunocytochemical methods (18). Antibodies directed against the CRALBP N-terminal 17 residues, C-terminal 31 residues, or a near N-terminal domain revealed a protein band with a slightly lower apparent mass in the D407 RPE cell extract compared with CRALBP in the human retinal extract or the purified recombinant control (Fig. 1). The strongest detection of CRALBP in the D407 RPE cell extract was obtained with the C-terminal antibody; no detection was apparent with the N-terminal antibody; and an intermediate level of detection was obtained with the third antibody. These results suggest that partial N-terminal proteolysis had occurred during protein extraction from the cultured cells as previously seen by Western analysis of CRALBP extracted from rat Müller cell cultures (28). These observations are consistent with the documented lability of the N terminus of CRALBP to proteolysis (20). In other Western analyses, the immunopositive bands could be competed away by excess purified recombinant CRALBP, but not by excess bovine serum albumin (data not shown). More intense immunodetection of CRALBP was apparent in the retinal extract than in the RPE cell culture extract; limited proteolysis was not apparent in the retinal extract. Reverse transcription-PCR using RNA from each of the three RPE cell culture systems followed by Southern blotting demonstrated the presence of CRALBP mRNA (data not shown). For these analyses, the PCR primers flanked intron 2 of RLBP1, and the Southern results distinguished CRALBP cDNA (241 bp) from genomic DNA (511 bp). Because the CRALBP N terminus is encoded within the reverse transcription-PCR product, these results also establish that the CRALBP mRNA in these RPE cells encodes an intact N terminus. Consistent with previous observations (17), greater apparent levels of CRALBP mRNA and protein were observed in nontransformed RPE cells grown on Matrigel or bovine corneal endothelial cell matrix than when grown on plastic. These results, together with the previous reports, establish that CRALBP is expressed in the three RPE cell culture systems used in this study.
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Regulation of RPE-specific Expression of CRALBP--
As a first
step toward identifying the elements in the CRALBP gene responsible for
its expression in the RPE, we transfected reporter gene constructs
containing 0.3-2.3 kb of the human CRALBP gene promoter region into
three human RPE cell model systems and into several human non-ocular
cell systems (Fig. 2). The results with
all three RPE cells were similar and showed that the 2.1- and 2.3-kb
CRALBP-reporter gene constructs direct high level luciferase expression
in the RPE cells, but not in the non-ocular cells. Also evident, the
0.3-kb CRALBP-reporter gene construct, but not the reverse orientation
(0.3R) construct, directs above background level luciferase expression
in the RPE (Fig. 2, C-E). This suggests that basal promoter
elements in RLBP1 exist between 243 and +80 bp. The
results also suggest that element(s) that enhance CRALBP expression in
RPE cells exist between
2089 and
1539 bp. None of the
CRALBP-reporter constructs directed significant levels of luciferase
expression in cultures of human liver or skin cells, although low
levels of nonspecific leaky expression were observed in cultures of
human breast cells (Fig. 2, F-H).
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Identification of PCE-1 as a Possible Positive Regulator in the
RPE--
The presence of two PCE-1 sequences in the 243 to +80
region was intriguing given that similar sequences have been implicated in the regulation of photoreceptor-specific gene expression (13, 15,
16). To investigate the possible role of CRALBP PCE-1, we prepared
mutant 0.3- and 2.1-kb reporter constructs with altered PCE-1 sites
(Fig. 3A) and performed
transient transfections with human D407 RPE cells. The transfection
results (Fig. 3B) show that altering the PCE-1 sites in the
2.1-kb construct decreased the luciferase reporter activity relative to
the 2.1-kb wild-type constructs. Reporter activity for the 2.1-kb
mutant constructs was reduced ~35% with either PCE-1 site altered
(pce-dM or pce-pM), ~60% with both PCE-1 sites mutated (pce-2M), and
>70% when PCE-1 and adjacent sequence were deleted and mutated
(pce-
M). Reporter activity for the 0.3-kb mutant constructs
approximated that observed for the 0.2-kb wild-type construct lacking
PCE sites. These results support a role for PCE-1 in regulating CRALBP
expression in the RPE.
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PCE in the CRALBP Proximal Promoter Interacts with Proteins in the Retina and RPE-- To follow up on the transient transfection results, EMSAs were performed to investigate possible DNA-protein interactions involving the CRALBP PCE region. EMSA with a 42-bp probe, designated PCE42 (Table I), demonstrated four shifted protein complexes with a bovine retinal nuclear extract (indicated by arrows in Fig. 4A). These complexes were not apparent in nuclear extract from rat seminal vesicle or human breast cells (Fig. 4A). The bovine retinal complexes were reduced in number and diminished in intensity by competition with excess unlabeled PCE42 probe (Fig. 4B). These complexes could be competed away with excess amounts of a structurally related probe (arrestin-42; Table I), but not with the unrelated clusterin probe (data not shown). These results suggest that the observed DNA-protein complexes are tissue- and sequence-specific.
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Southwestern Analysis of RPE Proteins Interacting with the CRALBP PCE-- Southwestern analysis was used to investigate the size and complexity of proteins in the D407 RPE nuclear extract that bind to the wild-type CRALBP 32P-labeled PCE42 probe. The results (Fig. 5) reveal major doublet bands with apparent molecular masses near 90-100 kDa and a less predominant lower band at ~31 kDa. Unlabeled probes that contain intact PCE sequences (i.e. PCE42, PCE29, bce-2M and arrestin-42) were found to be the most effective competitors of binding. Unlabeled probes with both PCE-1 sites altered (pce-2M) or with both PCE-1 sites and both BCE sites altered (p&b-2M) could not block binding of the wild-type PCE42 probe with RPE nuclear protein. These results are consistent with the EMSA results in Fig. 4 and further demonstrate a specific interaction between PCE and RPE nuclear proteins.
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DISCUSSION |
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CRALBP was first detected and purified from bovine retina ~20 years ago and subsequently found to be abundant in the RPE and to carry 11-cis-retinal and 11-cis-retinol as endogenous ligands in the RPE and retina (7). Structure-function studies have defined ligand stereoselectivity and photosensitivity and a topological and epitope map and established in vitro evidence for a substrate carrier function in the RPE (6, 7, 20). Human and bovine CRALBP cDNAs have been cloned, and human recombinant CRALBP has been produced and used in functional domain analyses (21, 29, 30). This study has explored the molecular mechanisms controlling expression of the human CRALBP gene (RLBP1) in the RPE.
The presence of photoreceptor consensus elements in genes other than
those expressed in photoreceptors suggests a broader tissue spectrum of
potential regulatory activity for PCE-1. We investigated the potential
role of PCE-1 in human CRALBP gene expression in three human RPE cell
culture systems. The 0.3-kb CRALBP-reporter construct, which contains
two PCE-1 sites, consistently generated above background level reporter
gene expression. None of the CRALBP gene constructs provided
significant reporter activity in three different non-ocular cell lines.
These results demonstrate that basal promoter function lies in the
sequences contained in the 0.3-kb construct (RLBP1 positions
243 to +80). The increase in reporter activity observed with the
2.1-kb construct compared with the 1.6-kb construct suggests the
presence of an enhancer element between
2089 and
1539 bp. Notably,
Alu repetitive sequence exists in the 1.6-kb construct
between
1438 and
1157 bp; whether this region influences CRALBP
gene expression remains to be determined. Given the consistent low
level reporter activity of the 0.8-kb construct, a repressor element
may exist within the
702 to
243 region. Reporter activity in RPE
cells for the 2.1-kb construct with either of the two PCE-1 sites
altered was significantly reduced relative to wild-type constructs and
even more dramatically reduced with both PCE-1 sites mutated or deleted
(Fig. 3). These data suggest a positive role for PCE-1 in regulating
CRALBP expression in the RPE. The apparent enhancer activity in
RLBP1 between
2089 and
1539 bp appears to be modulated
in part by DNA-binding protein(s) interacting with PCE-1 between
165
and
140 bp.
The nature of the RLBP1-protein interactions were probed by EMSA using a 42-bp wild-type probe (PCE42) from RLBP1 designed to mimic an arrestin probe used in other binding studies. The binding pattern obtained with PCE42 resembled that obtained with bovine retinal extract in a previous study (13). Two of the PCE42-protein interactions in the retinal extract appear to be specific based on competition results and the fact that similar complexes were not observed with nuclear extracts from seminal vesicle and breast cells (Fig. 4, A and B). The apparent low molecular mass complexes in extracts from seminal vesicle and breast cells are not present in retinal extracts. These complexes do not appear to be relevant to CRALBP gene expression in visual tissues; however, their significance was not explored. Shorter oligonucleotide probes (PCE29) were designed to more specifically evaluate RPE protein interactions with PCE-1 and the 6-bp BCE site adjacent to each PCE-1 site. Two RPE nuclear protein complexes were observed with the PCE29 probes, which require intact PCE-1 and BCE sequences (Fig. 4C). The results from competitive EMSA with mutant probes pce-2M and bce-2M suggest that PCE-1 mediates formation of both complexes and that PCE-1 and the BCE are involved in formation of the more retarded complex (Fig. 4D). Complex formation with PCE-1 appears to be required for the more retarded complex to form, encompassing the BCE (Fig. 4D). Overall, the results from the reporter gene assays and EMSA strongly support a role for PCE-1 in controlling CRALBP expression in the RPE. It is not yet clear whether PCE-1 and the BCE comprise a single cis-element or two separate elements.
Up to three RPE nuclear complexes were detected by Southwestern
analysis using the PCE42 probe from RLBP1. Further study is needed to determine whether these components are frayed or
post-translationally modified versions of one polypeptide, different
proteins, or perhaps multimeric complexes. Relative to the two
90-100-kDa complexes, less probe appears to bind to the 31-kDa
component, suggesting that the smaller component may be less abundant.
The smaller complex may be a unique protein, but it could also be a
DNA-binding domain fragment from a larger protein. Interestingly, the
homologous Ret-1 element in the opsin gene appears to bind a 40-kDa
retinal nuclear protein (25, 31), and the homologous T-1 element in
the transducin gene appears to bind a 90-kDa retinal nuclear protein
(32). While Ret-1 and T
-1 are thought to bind different proteins,
the apparent masses of their respective nuclear factors are within
SDS-PAGE experimental error of the RPE nuclear complexes observed in
the present analysis. Intact PCE-1 sequences were required to block
formation of the complexes observed by Southwestern analysis, further
emphasizing the specificity of the interaction between PCE-1 and RPE
nuclear proteins. Notably, a PCE-1-containing probe designed from the
mouse arrestin gene (Table I) and homologous to Ret-1 and T
-1 was
also able to compete for binding with the CRALBP PCE42 probe. These
observations raise the question of whether a common PCE-1-binding
protein may function in the RPE and photoreceptors and perhaps other
tissues.
In addition to PCE-1 in the proximal promoter, other PCE-like sequences
exist in RLBP1 at 1432 to
1426 bp and
1030 to
1024 bp. Sequences similar to other cis-acting elements
regulating opsin expression can also be found in the CRALBP promoter,
including Ret-2 (at 35-45 bp) (33), RER-FPIII (at
239 to
228 bp)
(34), RER-FPIV (at
2078 to
2059 bp and in the 2.1-kb reporter
construct) (34), and glass-like (at
2484 to
2465 bp)
(35). TATA and CCAAT basal promoter elements are not present, but a
transcription initiator (Inr) sequence exists at
5 to +5 bp (36).
Consensus Sp-1, AP-1, glucocorticoid-responsive elements, YY1,
insulin-responsive elements, STRD, and other common transcription
factor-binding sites can be found by computer analysis (11, 27). Except
for the two PCE-1 sites between
165 and
140 bp, the significance of
these other potential regulatory elements remains to be determined.
PCE-1 in the RLBP1 proximal promoter appears to contain
essential nucleotides for CRALBP expression in the RPE. As determined for the photoreceptor-specific genes opsin (16, 37, 38) and
interphotoreceptor retinoid-binding protein (15) and as suggested for
arrestin (14), the RLBP1 proximal promoter may determine the
cell specificity of CRALBP expression, whereas upstream elements may
govern other aspects such as the quantity and timing of expression.
Regulation of opsin and interphotoreceptor retinoid-binding protein
genes involves more distal upstream elements, including enhancer
elements (34, 37). Indeed, our in vitro results suggest that
enhancer elements exist in the RLBP1 distal region between 2089 and
1539 bp. The current work extends the spectrum of gene regulatory activity for PCE-1 from rod photoreceptor cells to the RPE.
However, the spectrum may be even broader since CRALBP is expressed in
several ocular cell types and in brain oligodendrocytes. Furthermore, a
recent transgenic mouse study suggested that the related Ret-1 element
may influence gene expression in brain (16). A growing number of
retinal transcription factors appear to be coexpressed in the central
nervous system (39), suggesting that a common PCE-1-binding protein for
RLBP1 may function in the retina, RPE, brain, and perhaps
other tissues. Future in vivo studies will be needed to more
directly test this hypothesis.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the assistance of the Adirondack Biomedical Research Institute staff, including P. Scotty Adams and Amy Moquin in DNA synthesis/sequencing and Valerie Oliver, Marina LaDuke, Alice Vera, and Robert Sanson in manuscript preparation. We also thank Drs. Martin Tenniswood and Finian Martin for useful discussions throughout the course of this work.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants EY06603, EY09769, EY01765, and EY05951; the Foundation Fighting Blindness; unrestricted funds from Research to Prevent Blindness, Inc.; and the Rebecca P. Moon, Charles M. Moon, Jr., and Dr. P. Thomas Manchester Research Fund. Preliminary reports of this work were presented at the 67th Annual Meeting of the Association for Research in Vision and Ophthalmology, Ft. Lauderdale, FL, May 14-19, 1995 (Kennedy, B. N., Goldflam, S., Chang, M. A., Hacket, S., Campochiaro, P., Davis, A., Zack, D. J., and Crabb, J. W. (1995) Invest. Ophthalmol. & Visual Sci. 36, S124 (Abstr. 608); Chang, M. A., Kennedy, B., Crabb, J. W., Hackett, S., Campochiaro, P., and Zack, D. J. (1995) Invest. Ophthalmol. & Visual. Sci. 36, S124 (Abstr. 609) and at the 68th Annual Meeting of the Association for Research in Vision and Ophthalmology, Ft. Lauderdale, FL, April 21-26, 1996 (Kennedy, B. N., Chang, M. A., Campochiaro, P., Zack, D. J., and Crabb, J. W. (1996) Invest. Ophthalmol. & Visual Sci. 37, S336 (Abstr. 1540).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ This work was submitted in partial fulfillment of the requirements for a doctoral degree in the Cell and Molecular Biology Program jointly administered by the University College Dublin (Dublin, Ireland) and the Adirondack Biomedical Research Institute (Lake Placid, NY).
Present address: Jules Stein Eye Inst., UCLA, Los Angeles, CA
90024.
Recipient of a career development award from Research to
Prevent Blindness, Inc.
§§ To whom correspondence should be addressed: Protein Chemistry Facility, Adirondack Biomedical Research Inst., 10 Old Barn Rd., Lake Placid, NY 12946. Tel.: 518-523-1281; Fax: 518-523-1849; E-mail: jcrabb{at}cell-science.org.
1 The abbreviations used are: RPE, retinal pigment epithelium; CRALBP, cellular retinaldehyde-binding protein; PCE, photoreceptor consensus element; BCE, binding CRALBP element; bp, base pair(s); kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift analysis; SW, Southwestern.
2 Available on the World Wide Web at http://bimas.dcrt.nih. gov:80/molbio/signal/.
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