Regulation of DSPP by OF45/MEPE and Characterization of the Human OF45/MEPE Gene Promoter

*1047 Regulation of DSPP by OF45/MEPE and Characterization of the Human OF45/MEPE Gene Promoter*
A. UNTERBRINK¹, M. MACDOUGALL¹, and S. CHEN², ¹ University of Texas Health Science Center at San Antonio, USA, ² UTHSCSA, San Antonio, TX, USA OF45, also known as MEPE, is a newly identified tumor-secreted protein in osteomalacia that is expressed in bone. Our laboratory has determined that OF45/MEPE is expressed by odontoblasts and ameloblasts. This gene maps to 4q21 within a dentin/bone gene cluster containing OPN, BSP, DMP1 and DSPP. Previous studies had determined that OF45/MEPE down-regulates DSPP (Unterbrink et al., 2002). Objectives: 1) Determine if OF45/MEPE regulates DSPP expression in dental pulp cells; 2) Define the promoter segment conferring OF45/MEPE regulation of DSPP; and 3) Characterize the human OF45/MEPE gene promoter. Methods: In vitro functional studies were performed testing the effects of MEPE over-expression on MD10-H1 dental pulp cells. Six 5' DSPP promoter luciferase constructs were generated and cloned into pGL3 luciferase (LUC) basic vector (Promega). These constructs contained –2664 nt, -1447 nt, -1243 nt, -791 nt, -510 nt and -95 nt of the DSPP upstream region and +54 nt of exon 1. The OF45/MEPE expression construct and the various DSPP report constructs were transiently co-transfected into MD10-A2 odontoblast cells and grown for 48 hrs. Cells were then harvested, LUC activity measured, and analyzed using an ANOVA. A ~1.2 kb segment of the human OF45/MEPE gene promoter was isolated, DNA sequenced, and analyzed for potential cis-elements using the computer programs TransFac and Signal Scan. Results: Over-expression of OF45/MEPE significantly down-regulate DSPP in dental pulp cells (p<0.05). Analysis of the DSPP promoter cut-backs showed that only the full length 2664 bp fragment showed significant regulation by OF45/MEPE (p<0.002). Several known cis-elements such as a TATA, and CAAT box were found in the OF45/MEPE promoter as well as for AP-2, AP-1, Pit-1, and TCF-1. Conclusions: This study demonstrated an OF45/MEPE cis-regulatory element is contained in a 1217 bp segment (between the -2664 bp and -1447 bp) of the mouse DSPP promoter. (NIDCR- F30-DE14326, AU).
Seq #134 - Dentin Matrix Proteins/Regulation 11:00 AM-12:15 PM, Friday, 14 March 2003 Henry B. Gonzalez Convention Center Exhibit Hall C
Back to the Mineralized Tissue Program Back to the 32nd Annual Meeting and Exhibition of the AADR (March 12-15, 2003)
Top Level Search