Molecular Regulation of Matrix Extracellular Phosphoglycoprotein Expression by Bone Morphogenetic Protein-2

Abstract

Matrix extracellular phosphoglycoprotein (MEPE) is mainly expressed in mineralizing tissues, and its C-terminal proteolytic cleavage product is an acidic-serine-asparate-rich-MEPE-associated motif (ASARM) that is a strong regulator of body phosphate metabolism and mineralization. There is sufficient data supporting a role for MEPE protein function in mineralization, however, little is known about the regulation of MEPE gene expression. As bone morphogenetic protein-2 (BMP-2) is one of the most important signals for calvarial mineralization and MEPE expression is higher in mineralized tissues, we attempted to uncover a regulatory circuit between BMP-2 and MEPE expression. Mepe expression is very low in proliferating MC3T3-E1 cells, but is dramatically increased in the mineralization stage and is strongly stimulated by treatment with BMP-2, even in proliferating cells. Overexpression and knockdown experiments of Smads, Dlx5, and Runx2 indicated that they are indispensable mediators of BMP-2-induced Mepe expression. In contrast, Msx2 showed strong inhibition of Mepe transcription. PHEX is an enzyme that prevents the release of the ASARM motif, a mineralization inhibitor, from the MEPE molecule. Thus, the MEPE/PHEX ratio may be a good indicator of mineralization progression because we found that the mRNA ratio and protein levels were low when osteoblasts were actively differentiating to the mineralization stage and the ratio was high when the cells reached the mineralization stage when it is assumed that osteocytes may protect themselves and make a space to survive from the mineralized matrix by releasing the ASARM motif. Collectively, MEPE expression is bone cell-specific and induced by the BMP-2 signaling pathway. In addition, the MEPE/PHEX ratio of the cell could be a very important barometer indicating the progression of tissue mineralization.Mineral homeostasis in the body is critical for healthy bones and teeth, and is generally regulated by the calcium-phosphate balance in the bone and kidney networks. In the past, the vitamin D/parathyroid hormone axis was assumed to be a single major circuit in bone-renal phosphate regulation, but recently, new bone-renal phosphate regulating factors have been identified. The finding of fibroblast growth factor 23 (FGF23), 2 phosphate-regulating genes with homologies to endopeptidases on the X chromosome (PHEX) and matrix extracellular phosphoglycoprotein (MEPE) genes, and their pathophysiological roles in the genetic diseases of mineral metabolism, have provided a great deal of insight into the understanding of bone-and mineral-related diseases. Among these, autosomal-dominant hypophosphatemic rickets (OMIM number 193100) is characterized by renal phosphate wasting, hypophosphatemia, and inappropriately normal 1,25-dihydroxyvitamin D 3 levels (1). Autosomal-dominant hypophosphatemic rickets is caused by a missense mutation of FGF23, which is resistant to proteolysis by PHEX and increases the half-life of full-length phosphaturic FGF23 (2). Second, X-linked hypophosphatemic...

The Canonical BMP Signaling Pathway Plays a Crucial Part in Stimulation of Dentin Sialophosphoprotein Expression by BMP-2

“…4D, lanes 7 and 14, asterisk). Contrary to the results with the Alp and Mepe promoters (7,14), Msx2 did not form a complex with the probes when Msx2 protein was increased, although it did inhibit Dlx5 binding to h2 (Fig. 4D).…”

“…2C). We previously reported that Dlx5 stimulates Alp and Mepe expression, but Msx2 antagonizes the stimulatory effect of Dlx5 by competing for binding to the same response elements in promoters (7,14). On the basis of these results, we hypothesized that Msx2 also suppresses Dspp expression.…”

“…They reported that BMP-2 induces Dspp expression through NF-Y, but they did not address the relationship between canonical BMP-2 signaling and Dspp expression. We have investigated the role of the canonical BMP-2 signaling pathway in expression of various bone marker genes and have identified Mepe as one gene whose expression is regulated by this pathway (7). Because BMP-2 is also important in odontoblast differentiation, and DSPP is involved in dentin mineralization as matrix extracellular phosphoglycoprotein is in bone mineralization, we hypothesized that Dspp expression is also regulated by the canonical BMP-2 signaling pathway.…”

“…The genes encoding these proteins are clustered on chromosome 4q21 in humans and 5q in mouse, and the proteins are commonly involved in dentin and bone mineralization and share an acidic serine-and aspartate-rich motif (7).…”

“…These double-stranded DNA probes were end-labeled with [␥-32 P]ATP (PerkinElmer Life Science) using a DNA 5Ј End Labeling System (Promega). 5Ј-DNA end labeling and EMSA were performed in accordance with the manufacturer's instructions and our previous papers (7,25). The Dlx5 and Msx2 proteins were produced by in vitro transcription and translation using TNT-coupled Reticulocyte Lysate (Promega).…”