Transcriptional regulation mechanism of mineralization controlling factors, MEPE and DSPP

Abstract

Mineralization is important process in the hard tissue including teeth and bone. Hypermineralization or hypomineralization causes malfunction, and then inhibits normal hard tissue formation. Matrix Extracellular PhosphoglycoprotEin (MEPE) and Dentin SialoPhosphoProtein (DSPP) are well known for potent factors in the bone and dentin mineralization, separately. They are members of Small Integrin Binding LIgand, N-linked Glycoprotein (SIBLING) family protein, which is composed of MEPE, DSPP, bone sialoprotein (BSP), osteopontin (OPN) and dentin matrix protein 1 (DMP1), and clustered on chromosome 4q21 in human and 5q in mouse, and have a similarity to produce Acidic-Serine-Asparate-Rich-MEPE-associated motif (ASARM). There are sufficient data supporting a role for MEPE protein function in mineralization, however, little is known about the regulation of MEPE gene expression. Therefore, we tried to find the transcriptional regulation mechanism of MEPE. Since MEPE expression is specifically expressed in the osteoblast, osteocyte, and BMPs, Wnts are well known by the importance in the bone formation, we could suppose the relationship in the MEPE expression through BMPs and Wnts. Mepe expression was increased by BMP-2 or Wnt3a treatment in the MC3T3-E1 cells, and conserved homeodomain (Dlx/Msx) response elements and LEF-1 binding elements were in the MEPE promoter region. First, in the part I, I demonstrated that MEPE expression is regulated by canonical BMP-2 signaling pathway through BMP R-Smads, Dlx5, Msx2 and Runx2 with Real time PCR, luciferase reporter assay with promoter deletion/mutant reporter vectors, EMSA and ChIP assay. Second, in the part II, I showed that MEPE expression is also regulated by canonical Wnt3a signaling pathway through -catenin/LEF1. Furthermore, I found that Wnt3a makes BMP-2 autocrine loop, and stimulates MEPE expression indirectly through BMP-2. Based on these MEPE gene transcriptional regulation mechanism, I tried to know more about MEPE function with PHEX in the bone formation and mineralization. In the part III, I demonstrated that DSPP expression is regulated by canonical BMP-2 signaling pathway in a same manner of the MEPE expression regulation by BMP-2. Transcriptional regulation mechanism had been identified through Runx2, CCAAT binding heterotrimeric transcription factor Y (NF-Y) and Twist1. Canonical BMP signaling pathway has been known to participate greatly in biomineralization, and DSPP expression is stimulated by BMP-2 treatment, however, previous studies overlooked the involvement of canonical BMP signaling pathway in the Dspp expression. Therefore, this study was designed to uncover this question, and I showed that DSPP expression is regulated by canonical BMP-2 signaling pathway. Collectively, I demonstrate here that BMP-2 and Wnt3a stimulate MEPE expression directly, and BMP-2 autocrine loop by Wnt3a also regulates MEPE expression indirectly in the MC3T3-E1 preosteoblastic cells. In addition to the MEPE transcriptional regulation mechanism, I suggest that MEPE/PHEX ratio is important to control bone formation and mineralization. Furthermore, I also proved that DSPP expression is canonical BMP-2 signaling pathway in the MDPC-23 preodontoblastic cells.