The Gene Encoding Human Cellular Retinol Binding Protein 1 (hCRBP1) Is Negatively Regulated by the Transcription Factor NF1 in the ARPE19 Cell Line

Abstract

Abstract: : Purpose: Retinol (ROL) is essential for growth, development, reproduction and vision. Because of its hydrophobic nature, ROL needs to bind cellular retinol–binding protein 1 (CRBP1) for its transportation through the cytosol. In the eye, CRBP1 expression was found to be high in the retinal pigment epithelium (RPE) in which it plays a major role in the migration of all–trans retinol from the photoreceptors to the RPE. Previous works conducted in our laboratory identified seven target sites (Fp1 to Fp7) protected from DnaseI digestion by yet unknown nuclear transcription factors along the promoter of the hCRBP1 gene. The Fp1 element was shown to bear putative target sites for members of the NF1 (two site: NF1A and NF1B) and Sp1 (one site) families of transcription factors. In the present study, we investigated the respective regulatory influence of the Sp1 and NF1 binding sites in primary cultures of rabbit corneal epithelial cells (RCECs) and the human RPE–derived cell–line ARPE–19. Methods: The regulatory influence of the Fp1 element was examined by transfection of both RCECs and ARPE19 cells with recombinant constructs bearing various lengths from the endogenous hCRBP1 promoter fused to the CAT gene. The ability of the Fp1 element to regulate other gene’s promoter was assessed by the transfection of plasmids bearing Fp1 (or mutated derivatives of Fp1) inserted upstream the basal promoter of the mouse p12 gene. Both the expression and DNA binding properties of Sp1/Sp3 and NF1 were monitored by EMSAs in RCECs and ARPE19 cells grown to mid– and post–confluence. Results: Deletion of Fp1 in the hCRBP1 promoter increased promoter function upon transfection into cultured cells. Consistent with these results, insertion of Fp1 upstream the unrelated p12 promoter yielded a dramatic repression in all transfected cells. Site–directed mutation of FP1 indicated that NF1A, but not NF1B, accounts for this repression. Supershift experiments provided evidence that neither Sp1 nor Sp3 can bind the putative Sp1 site from Fp1. Conclusions: These results suggest that Fp1 functions as a ubiquitous, negative regulatory element (hCRBP1 or p12). Both Sp1 and Sp3 positively modulated the activity of the hCRBP1 promoter but through the use of regulatory sequences located between positions –223 to +69 that are deleted of both the Fp1 and Fp5 elements.

Keywords: gene/expression • transcription factors • retinal pigment epithelium