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Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones. Although expression of this gene has been demonstrated in multiple tissues, its regulation is not well understood. Our previous studies on the roles of the retinoblastoma protein (pRB) and p300͞CBP in the regulation of osteoblast differentiation revealed a link between osteopontin induction and the synthesis of alkaline phosphatase. In this paper, we describe results specifically linking induction of osteopontin to the enzymatic activity of alkaline phosphatase in the medium, which results in the generation of free phosphate. This elevation of free phosphate in the medium is sufficient to signal induction of osteopontin RNA and protein. The strong and specific induction of osteopontin in direct response to increased phosphate levels provides a mechanism to explain how expression of this product is normally regulated in bone and suggests how it may become up-regulated in damaged tissue. A s cells undergo terminal differentiation, various markers are induced in an ordered and sequential manner, but required steps in the induction process are not always clear because individual events in the sequence are not easily separable. The biology of the DNA tumor virus oncogene, adenovirus E1A, points to the cellular E1A targets, retinoblastoma protein (pRB) and p300͞CBP, as important regulators of gene expression during terminal differentiation (1-4). We have taken advantage of E1A genetics to explore the roles of the pRB and p300͞CBP protein families in expression of early and late markers during osteoblast differentiation. The MC3T3-E1 cell line is derived from newborn mice calvaria (5). It is an established cell line, but the cells maintain much of the tightly linked controls between proliferation and differentiation that usually are seen only in primary cells (6). Treatment with ascorbic acid stimulates these cells to differentiate along the osteoblast line (6-8). Induced cells deposit a collagenous extracellular matrix, accompanied by the activation of specific genes associated with the osteoblast phenotype, such as alkaline phosphatase, osteocalcin, and osteopontin. If a source of organic phosphate such as ␤-glycerol phosphate is present, a discrete zone of hydroxyapatitecontaining mineral is formed within the collagen fibrils. The sequence from induction to mineralization proceeds in a tightly regulated order over a span of 2 to 3 weeks (see schematic in Fig. 1), which permits a detailed analysis of the order of events.We previously have characterized two MC3T3-E1 cell lines stably express...

The spatial and temporal coordination of the many events required for osteogenic cells to create a mineralized matrix are only partially understood. The complexity of this process, and the nature of the final product, demand that these cells have mechanisms to carefully monitor events in the extracellular environment and have the ability to respond through cellular and molecular changes. The generation of inorganic phosphate during the process of differentiation may be one such signal. In addition to the requirement of inorganic phosphate as a component of hydroxyapatite mineral, Ca(10)(PO(4))(6)(OH)(2), a number of studies have also suggested it is required in the events preceding mineralization. However, contrasting results, physiological relevance, and the lack of a clear mechanism(s) have created some debate as to the significance of elevated phosphate in the differentiation process. More recently, a number of studies have begun to shed light on possible cellular and molecular consequences of elevated intracellular inorganic phosphate. These results suggest a model in which the generation of inorganic phosphate during osteoblast differentiation may in and of itself represent a signal capable of facilitating the temporal coordination of expression and regulation of multiple factors necessary for mineralization. The regulation of protein function and gene expression by elevated inorganic phosphate during osteoblast differentiation may represent a mechanism by which mineralizing cells monitor and respond to the changing extracellular environment.

Bone is a dynamic tissue that undergoes renewal throughout life by a process whereby osteoclasts resorb worn bone and osteoblasts synthesize new bone. Imbalances in bone turnover lead to bone loss and development of osteoporosis and ultimately fracture, a debilitating condition with high morbidity and mortality. Silica is a ubiquitous biocontaminant that is considered to have high biocompatibility. We report that silica nanoparticles mediate potent inhibitory effects on osteoclasts and stimulatory effects on osteoblasts in vitro. The mechanism of bioactivity is a consequence of an intrinsic capacity to antagonize activation of NF-κB, a signal transduction pathway required for osteoclastic bone resorption, but inhibitory to osteoblastic bone formation. We further demonstrate that silica nanoparticles promote a significant enhancement of bone mineral density (BMD) in mice in vivo providing a proof of principle for the potential application of silica nanoparticles as a pharmacological agent to enhance BMD and protect against bone fracture.

We are using viral oncogene probes to study the pathways by which osteoblast-specific gene expression is induced in ascorbic acid-treated MC3T3-E1 cells. The 12S product of the adenovirus E1A gene binds directly to key cellular regulators and, as a result, represses tissue specific gene expression and blocks differentiation in a wide variety of cell types. The main cellular targets of the E1A 12S product are the pRB family and p300/CBP family. The p300 family appears to be the primary target for E1A-mediated repression of tissue-specific gene expression in a variety of cell types. We have generated MC3T3-E1 cell lines that stably express either the wild-type 12S product or a mutant that targets p300/CBP, but not the pRB family. Using these constructs to dissect osteoblast differentiation, we found that targeting of p300/CBP appears to be sufficient to repress alkaline phosphatase expression, although a low but functional level of expression can be maintained if the pRB family is not targeted as well. Induction of alkaline phosphatase expression and activity can be dissociated from expression of late-stage markers such as osteocalcin and osteopontin. Surprisingly, cell lines exhibiting severe repression of alkaline phosphatase activity differentiate to a mineral-secreting phenotype much like normal MC3T3-E1 cells. Osteopontin induction is dependent on at least a minimal level of alkaline phosphatase activity, although it is not dependent on induction of alkaline phosphatase at the RNA level. If alkaline phosphatase is supplied exogenously, osteopontin expression can be induced in conditions in which endogenous alkaline phosphatase is severely repressed.

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Many key processes central to bone formation and homeostasis require the involvement of osteoblasts, cells responsible for accumulation and mineralization of the extracellular matrix (ECM). During this complex and only partially understood process, osteoblasts generate and secrete matrix vesicles (MVs) into the ECM to initiate mineralization. Although they are considered an important component of mineralization process, MVs still remain a mystery. To better understand their function and biogenesis, a proteomic analysis of MVs has been conducted. MVs were harvested by two sample preparation approaches and mass spectrometry was utilized for protein identification. A total of 133 proteins were identified in common from the two MV preparations, among which were previously known proteins, such as annexins and peptidases, along with many novel proteins including a variety of enzymes, osteoblast-specific factors, ion channels, and signal transduction molecules, such as 14-3-3 family members and Rab-related proteins. To compare the proteome of MV with that of the ECM we conducted a large-scale proteomic analysis of collagenase digested mineralizing osteoblast matrix. This analysis resulted in the identification of 1,327 unique proteins. A comparison of the proteins identified from the two MV preparations with the ECM analysis revealed 83 unique, non-redundant proteins identified in all three samples. This investigation represents the first systematic proteomic analysis of MVs and provides insights into both the function and origin of these important mineralization-regulating vesicles.