In Vitro Effects of Dentin Matrix Protein-1 on Hydroxyapatite Formation Provide Insights into in Vivo Functions

Abstract

Dentin matrix protein-1 (DMP1) is a mineralized tissue matrix protein synthesized by osteoblasts, hypertrophic chondrocytes, and ameloblasts as well as odontoblasts. DMP1 is believed to have multiple in vivo functions, acting both as a signaling molecule and a regulator of biomineralization. Using a cell-free system in vitro, we evaluated the action of DMP1 in the regulation of hydroxylapatite (HA) formation and crystal growth. The non-phosphorylated recombinant protein acted as an HA nucleator, increasing the amount of mineral formed in a gelatin gel HA growth system relative to protein-free controls. The recombinant protein phosphorylated in vitro had no detectable effect on HA formation and growth. In contrast, phosphorylated bovine DMP1 expressed in marrow stromal cells with an adenovirus vector containing 29.7 phosphates/mol was an effective inhibitor of HA formation and growth. The native full-length protein appeared to be absent or present in only small amounts in the extracellular matrix of bones and teeth. However, two highly phosphorylated fragments representing the N-and C-terminal portions of DMP1 have been identified, apparently arising from proteolytic cleavage of four X-Asp bonds. The highly phosphorylated C-terminal 57-kDa fragment (containing 42 phosphates/mol), like the non-phosphorylated DMP1, was an HA nucleator. These data suggest that, in its native form, DMP1 inhibits mineralization, but when cleaved or dephosphorylated, it initiates mineralization. These in vitro data are consistent with the findings in the DMP1 knockout mouse.Dentin matrix protein-1 (DMP1) 1 is an acidic, phosphorylated, integrin-binding extracellular matrix protein first identified by screening a rat cDNA library (1). Northern blot analysis in the rat originally suggested that the DMP1 message was odontoblast-specific, with in situ hybridization showing DMP1 mRNA expression to be restricted to those fully differentiated odontoblasts engaged in active dentin matrix formation, with transient expression by ameloblasts (1, 2). More recent in situ hybridization studies show a much broader pattern of expression of DMP1, with expression associated with a number of mineralizing tissues, including bone and cementum (3). In rat and chicken bone, immunohistochemical detection showed an association of DMP1 with osteocytes, but not with osteoblasts (4). In the mouse, hypertrophic chondrocytes express more DMP1 than other cell types (5). DMP1 was detected at high levels by Northern analysis in fetal bovine brain and cultured long bone as well as in odontoblasts (6). Most recently, DMP1 was localized by immunohistochemistry in human lung cancer tissue (7).DMP1 is a small protein that has been postulated to have a high affinity for hydroxylapatite (HA) (8,9). This coupled with its localization in mineralized tissues suggested that DMP1 could have an important role in mineralization. DMP1 is a member of the SIBLING (small integrin-binding ligand, Nlinked glycoprotein) family of proteins (10, 11). Like other members of the SIBLING family...

Identification of Full-Length Dentin Matrix Protein 1 in Dentin and Bone

“…At present, it is unclear whether the trace amounts of full-length DMP1 secreted into the ECM have a function(s) different from that of its processed fragments. Previous in vitro mineralization studies showed that full-length bovine DMP1 made by bone marrow stromal fibroblasts inhibits hydroxyapatite formation whereas the COOH-terminal fragment isolated from rat bone promotes formation of the apatite crystals [14]. Future in vivo studies are warranted to test the biological functions of the full-length form of DMP1 in comparison with those of its processed fragments.…”

“…Based on the obvious differences in their biochemical features, it is logical to hypothesize that these variants may have distinct functions and play different roles in biomineralization. In vitro mineralization studies have demonstrated that the COOH-terminal fragment promotes mineralization by acting as a nucleator for hydroxyapatite formation [14][15][16]. Information regarding the biological functions of the NH 2 -terminal fragment and DMP1-PG is lacking.…”

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

“…We hypothesized that specific proteins were phosphorylated and dephosphorylated during the stages of initiation and proliferation of mineral crystals based on solution free studies of the effects of these proteins [2][3][4][5][6] and on our unpublished micro-array results showing upregulation of kinases during early stages of mineralization and down-regulation of phosphatases at the same time. We also hypothesized that the functions of the phosphatases was not simply to elevate the local inorganic phosphate concentration.…”

“…In cell-free mineralization studies the ability of extracellular matrix phosphoproteins such as osteopontin (OPN) [1,2], bone sialoprotein (BSP) [3,4], dentin matrix protein-1 (DMP1) [5] and phosphophoryn [6] to initiate hydroxyapatite (HA) mineral deposition and regulate the rate and extent of growth of the HA crystals is dependent on their state of phosphorylation. While the importance of the phosphate ion for appropriate mineral deposition is well recognized [7], it is only in recent years that attention has been paid to whether or not the extracellular matrix proteins were phosphorylated, as well as to their extent of phosphorylation and the enzymes involved in these posttranslational modifications [1][2][3][4][5][6][7][8][9].…”

“…While the importance of the phosphate ion for appropriate mineral deposition is well recognized [7], it is only in recent years that attention has been paid to whether or not the extracellular matrix proteins were phosphorylated, as well as to their extent of phosphorylation and the enzymes involved in these posttranslational modifications [1][2][3][4][5][6][7][8][9]. The importance of phosphorylation and dephosphorylation has been suggested by the abnormal patterns of mineralization in mutant animals with defective phosphoprotein degradative enzymes [10][11][12], defective phosphate transport [13,14], or defective phosphorylation [15].…”