Endoplasmic Reticulum Chaperone Protein GRP-78 Mediates Endocytosis of Dentin Matrix Protein 1

Abstract

Dentin matrix protein 1 (DMP1), a phosphorylated protein present in the mineral phase of both vertebrates and invertebrates, is a key regulatory protein during biogenic formation of mineral deposits. Previously we showed that DMP1 is localized in the nuclear compartment of preosteoblasts and preodontoblasts. In the nucleus DMP1 might play an important role in the regulation of genes that control osteoblast or odontoblast differentiation. Here, we show that cellular uptake of DMP1 occurs through endocytosis. Interestingly, this process is initiated by DMP1 binding to the glucose-regulated protein-78 (GRP-78) localized on the plasma membrane of preodontoblast cells. Binding of DMP1 to GRP-78 receptor was determined to be specific and saturable with a binding dissociation constant K D ‫؍‬ 85 nM. We further depict a road map for the endocytosed DMP1 and demonstrate that the internalization is mediated primarily by caveolae and that the vesicles containing DMP1 are routed to the nucleus along microtubules. Immunohistochemical analysis and binding studies performed with biotin-labeled DMP1 confirm spatial co-localization of DMP1 and GRP-78 in the preodontoblasts of a developing mouse molar. Co-localization of DMP1 with GRP-78 was also observed in T4-4 preodontoblast cells, dental pulp stem cells, and primary preodontoblasts. By small interfering RNA techniques, we demonstrate that the receptor for DMP1 is GRP-78. Therefore, binding of DMP1 with GRP-78 receptor might be an important mechanism by which DMP1 is internalized and transported to the nucleus during bone and tooth development.Acidic noncollagenous proteins play a pivotal role during biomineral formation. Dentin matrix protein 1 (DMP1) 2 was the first of the acidic proteins cloned from the dentin matrix.Although initially isolated from the dentin matrix and thought to be unique to dentin and named accordingly, DMP1 has now been found to be present in all mineralized tissues of the vertebrate system (1-3).We previously showed that DMP1 may act as a transcriptional regulator as DMP1 is localized in the nucleus of preosteoblasts (2). In the nucleus DMP1 played a regulatory role in the regulation of specific genes that control osteoblast and odontoblast differentiation (2, 3). Specifically, we showed that DMP1 functions as a transcriptional regulator of the odontoblast specific gene dentin sialophosphoprotein (3). The export of DMP1 from the nucleus during maturation of osteoblasts was found to be in response to a stimulus from calcium ions. In the extracellular matrix DMP1 can nucleate the formation and growth of hydroxyapatite (4, 5). Initial observations from the DMP1-deficient mouse showed no apparent skeletal or tooth phenotype during early development, suggesting that DMP1 function may be redundant. However, postnatal DMP1-null mice developed tooth formation defects, characterized by a partial failure of maturation from predentin to dentin (6). Collectively, these data indicate that DMP1 may play a pivotal role in regulating mineralized matrix formation. In ...

Stress Chaperone GRP-78 Functions in Mineralized Matrix Formation

“…We recently showed that DMP1 is endocytosed by pre-odontoblasts and DPSCs (dental pulp stem cells) by binding to GRP-78 receptor and is translocated to the nucleus (25). Although odontoblasts and osteoblasts possess similar characteristics, differences exist between the two cell types.…”

“…FITC labeling was carried out as published previously (25). The cells were then washed extensively, fixed, permeabilized, and mounted on glass slides using mounting media with DAPI nuclear stain (Vector Laboratories).…”

“…The pulldown experiments were carried out as previously published (25). Briefly, the membrane protein lysate was passed through a GST-DMP1 column.…”

“…In Vitro Binding Experiment-The in vitro binding of recombinant DMP1 to MC3T3 cells was carried out as published previously (25).…”

“…Involvement of Caveolae in the Endocytic Process Mediated by GRP-78-We previously showed that endocytosis of DMP1 occurs via the caveolar endocytic pathway in odontoblasts (25). Therefore, we investigated if the same endocytic route was adopted in osteoblasts.…”