The osteocyte, a terminally differentiated cell comprising 90%-95% of all bone cells 1,2 , may have multiple functions, including acting as a mechanosensor in bone (re)modeling 3 . Dentin matrix protein 1 (encoded by DMP1) is highly expressed in osteocytes 4 and, when deleted in mice, results in a hypomineralized bone phenotype 5 . We investigated the potential for this gene not only to direct skeletal mineralization but also to regulate phosphate (P i ) homeostasis. Both Dmp1-null mice and individuals with a newly identified disorder, autosomal recessive hypophosphatemic rickets, manifest rickets and osteomalacia with isolated renal phosphate-wasting associated with elevated fibroblast growth factor 23 (FGF23) levels and normocalciuria. Mutational analyses showed that autosomal recessive hypophosphatemic rickets family carried a mutation affecting the DMP1 start codon, and a second family carried a 7-bp deletion disrupting the highly conserved DMP1 C terminus. Mechanistic studies using Dmp1-null mice demonstrated that absence of DMP1 results in defective osteocyte maturation and increased FGF23 expression, leading to pathological changes in bone mineralization. Our findings suggest a bone-renal axis that is central to guiding proper mineral metabolism.Human disorders of phosphate (P i ) handling and skeletal mineralization can result from mutations in PHEX 6 , which cause X-linked hypophosphatemic rickets (XLH). A similar phenotype is also observed in Hyp mice, which have mutant Phex 7 and show increased osteocyte expression of the phosphaturic factor FGF23 (ref. 8 Individuals F1-1 and F1-3 presented with rickets and progressive lower limb deformity in late infancy, whereas sister F1-2 had rachitic changes on a chest X-ray at age 7 months. In contrast, F2-1 presented with a mild genu valgum at 8 years of age. The pre-or off-treatment age-related metabolic profiles for both kindreds were similar, characterized by hypophosphatemia owing to renal phosphate-wasting (serum P i : 0.7-0.9 mmol/l, normal: 1.2-1.8; threshold maximum for renal tubular phosphate reabsorption/glomerular filtration rate (TmP/GFR): 0.61-0.81 mmol/l, lower limit of normal: ≥1.0), high normal to moderately elevated alkaline phosphatase, normal intact parathyroid hormone (PTH) levels (4.6-6.9 pmol/l, normal: 1.6-6.9), normocalcemia (ionized calcium: 1. Resolution of rickets and normalization of alkaline phosphatase were observed during treatment with phosphate supplementation and calcitriol; however, the TmP/GFR remained low. Linear growth trajectories were heterogeneous among the affected individuals: affected individuals in F1 had a mid-parental height of 154.5 cm (5 th -10 th percentiles), with F1-1 and F1-2 measuring 153 cm (5 th percentile) and 136.5 cm (<5 th percentile) at final adult height, respectively. F1-3 had a height of 153.5 cm at 10 months post-menarche, well within the genetic target. The affected individual in F-2 had a final adult height of 172 cm (90 th -95 th percentile), 3 cm above the upper limit of her genetic ta...

Odontoblasts in dentin and osteocytes in bone contain dendritic processes. To test if their dendrites share a common feature, we compared their cellular morphology as visualized using scanning electron microscopy. Analysis of our data showed that both cells share an identical dendritic canalicular system and express extensive processes forming a complex network within the mineralized matrix. Because dentin matrix protein 1 (DMP1), an extracellular matrix protein, is highly expressed in both types of cells, we next tested, using a transgenic approach, whether a 9.6-kb Dmp1 promoter-4-kb 1st intron would be able to target Cre cDNA in these cells for expression/deletion of other genes in odontoblasts and osteocytes. We determined the specificity and efficiency of Cre activity by crossing Dmp1-Cre mice with ROSA26 reporter mice. Results showed that odontoblasts and osteocytes were specifically targeted, suggesting that this animal model will be useful for the preferential study of gene functions in both types of cells.

The dentin matrix protein-1 (DMP-1) gene is identified in odontoblasts during both embryonic and postnatal development. In vitro study suggests that this noncollagen acidic phosphoprotein plays a role in mineralization. However, deletion of the Dmp-1 gene has little effect on tooth development during embryogenesis. To address the role of DMP-1 in tooth during postnatal development, we analyzed changes of dentinogenesis in Dmp-1 null mice from 3 days after birth to 1 year. Here we show that Dmp-1 null mice postnatally develop a profound tooth phenotype characterized by a partial failure of maturation of predentin into dentin, enlarged pulp chambers, increased width of predentin zone with reduced dentin wall, and hypomineralization. The tooth phenotype of these mice is strikingly similar to that in dentin sialophosphoprotein (Dspp) null mice and shares some features of the human disease dentinogenesis imperfecta III. We have also demonstrated that DSPP levels are reduced in Dmp-1 null mice, suggesting that DSPP is probably regulated by DMP-1 during dentinogenesis. Finally, we show the absence or delayed development of the third molar in Dmp-1 null mice, which is probably secondary to defects in Dmp-1 null bone. Taken together, these studies suggest that DMP-1 is essential for later dentinogenesis during postnatal development.Dentin is a mineralized tissue that closely resembles bone in composition and mechanism of formation. The mechanisms for mineralization are largely unclear, although two hypotheses are proposed to explain initiating mineralization: matrix vesicles in mantle dentin and collagen-phosphophoryn complexes in circumpulpal dentin (1). The in vitro studies also suggest that phosphorylated extracellular matrix (ECM) 1 proteins localized within collagen gap zones can bind calcium and phosphate ions in an appropriate conformation to nucleate the formation of apatite crystals (2, 3). One of the noncollagenous proteins that appears to play an important role in dentin ECM formation and mineralization is dentin matrix protein-1 (DMP-1).DMP-1, an acidic phosphorylated extracellular matrix protein (4), is expressed in odontoblasts that secrete matrix proteins to form dentin. Using protein chemistry approaches, some progress has been made in identifying the normally processed forms of DMP-1 in mineralized tissues (5). Although full-length DMP-1 has been cloned and sequenced, the corresponding intact protein has not been isolated from mineralized tissues. However, two proteolytic fragments, a 37-kDa N-terminal fragment and a 57-kDa C-terminal fragment, have been isolated from bone and dentin extracts (5). Recent studies suggest that DMP-1 can be cleaved by bone morphogenetic protein-1/tolloidlike proteinases (6).In vitro studies suggest that overexpression of Dmp-1 induces differentiation of mesenchymal cells to odontoblast-like cells and enhances mineralization (7) and that DMP-1 can bind to Ca 2ϩ and initiate mineral deposition in vitro (8). However, effects of recombinant DMP-1 on in vitro mineralization are ...

Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.