Stress Chaperone GRP-78 Functions in Mineralized Matrix Formation

Abstract

Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78. GRP-78,2 also known as hspa5, is a member of the heat shock protein 70 family. Its primary function is to act as a molecular chaperone in the endoplasmic reticulum (ER) and facilitates the proper folding and assembly of membrane and secretory proteins. However, GRP-78 has been shown to be up-regulated under stress conditions such as the presence of toxic agents (1-4). GRP-78 has been reported to be translocated to the detergent-resistant fractions of the plasma membrane of these cells (5, 6). It has also been implicated to function as a receptor for ␣2-macroglobulin and different viruses through lipid rafts (7-9). Interestingly, GRP-78 has been shown to possess calcium binding properties with reports of calcium binding affinity ranging from 1-2 mol of calcium/mol of GRP-78 to much larger quantities (10). The biological significance of the calcium binding ability of GRP-78 is, however, a subject of debate, with its role primarily being attributed to binding of ER Ca 2ϩ similar to other sequestering proteins.Recent studies have shown that several stress proteins play a significant role in osteoblast differentiation. Specifically, Old Astrocyte Specifically Induced Substance, an ER stress transducer, has been implicated to be involved in bone formation (11). Additionally, a study on the analysis of the secretome of differentiating human mesenchymal stem cells showed an increase in the amount of secreted stress proteins (12). This study quantitatively analyzed the presence of GRP-78 in the secreted pool of proteins and showed that the amount of secreted protein increased when the cells were subjected to in vitro differentiation. Increase in expression of GRP-78 was attributed to...

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“…Further, we have shown that GRP78 secretion also increased upon induction of osteogenic or odontogenic differentiation of respective precursor mesenchymal cells (63). In vitro binding experiments indicated that GRP78 binds to type I collagen and DMPl.…”

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