Transcriptional Regulation of Cellular Retinaldehyde-binding Protein in the Retinal Pigment Epithelium

Abstract

Cellular retinaldehyde-binding protein (CRALBP) is abundantly expressed in the retinal pigment epithelium (RPE) and Mü ller cells of the retina, where it is thoughtto 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. 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...

Both PCE-1/RX and OTX/CRX Interactions Are Necessary for Photoreceptor-specific Gene Expression

“…Our results are also in agreement with those of Yu et al (21), who showed that in transgenic mice carrying four tandem copies of the PCE-1/Ret1 element fused to the lacZ reporter gene, ␤-gal was expressed not only in photoreceptor cells, but also in the ganglion cell layer and ciliary epithelium. The PCE-1 element in the 5Ј-flanking region of cellular retinaldehyde-binding protein may play a important role in the expression of cellular retinaldehyde-binding protein in RPE cells (22). In addition, present supershift analysis suggested that the PCE-1 site bound to RX as well as some other factor(s).…”

Transcription Factor SOX9 Plays a Key Role in the Regulation of Visual Cycle Gene Expression in the Retinal Pigment Epithelium

“…The retinal pigment epithelium (RPE) 4 is essential for supporting the survival and function of retinal photoreceptor cells (1). One of the key functions of the RPE is enzymatic regeneration of the photoreceptor visual chromophore (11-cis-retinal) through the visual cycle, a cyclical pathway consisting of a series of enzymatic reactions in which each step converts retinoid intermediates into a substrate for the next step (2).…”

“…Major RPE-expressed components of the visual cycle are three enzymes (RPE65, LRAT, and RDH5), two retinoid carrier proteins (RLBP1 and RBP1), and a modulator (RGR), which work sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptors. Although efficient functioning of the visual cycle requires coordinated expression of its various components and some progress has been made in defining the cis-regulatory elements involved in transcriptional regulation of individual visual cycle genes such as Rpe65 and Rlbp1 (3)(4)(5)(6)(7), only limited studies have addressed the regulatory mechanisms that coordinate expression across the visual cycle genes.…”

Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation

“…The human RPE65 5Ј-upstream region has also been studied using sequence analyses, cell transfection assays, and DNase I footprinting (24). Analysis of the 5Ј-up-stream region of the human cellular 11-cis-retinaldehyde-binding protein gene (RLBP1), which is preferentially expressed in RPE and Muller cells, suggested that a photoreceptor consensus element-1 (PCE-1, CAATTAG) located in the proximal promoter region might act as a positive regulator of RPE gene expression (25). This is interesting because PCE-1 was generally considered a photoreceptor element, and a sequence similar to PCE-1 is also present within the proximal promoter region of both human and murine Rpe65; however, its biological relevance has not yet been experimentally tested.…”