Although considerable advances in our understanding of the mechanisms of phosphate homeostasis and skeleton mineralization have recently been made, little is known about the initial events involving the detection of changes in the phosphate serum concentrations and the subsequent downstream regulation cascade. Recent data has strengthened a long-established hypothesis that a phosphate-sensing mechanism may be present in various organs. Such a phosphate sensor would detect changes in serum or local phosphate concentration and would inform the body, the local environment, or the individual cell. This suggests that phosphate in itself could represent a signal regulating multiple factors necessary for diverse biological processes such as bone or vascular calcification. This review summarizes findings supporting the possibility that phosphate represents a signaling molecule, particularly in bone and cartilage, but also in other tissues. The involvement of various signaling pathways (ERK1/2), transcription factors (Fra-1, Runx2) and phosphate transporters (PiT1, PiT2) is discussed.

Inorganic phosphate (Pi) and the matrix Gla protein (MGP) are key regulators of bone formation. We have recently shown that Pi upregulates MGP in growth plate chondrocytes, which may represent a negative feedback loop for the control of mineralization. Osteoblasts from Fra-1-deleted mice express low levels of MGP, whereas the expression of MGP is elevated in Fra-1 transgenic osteoblasts, suggesting a role for Fra-1 in MGP expression and bone formation. In this study, we aimed at deciphering the relationships between Pi and MGP in osteoblasts to determine the molecular mechanisms involved in the Pi-dependent regulation of MGP. In MC3T3-E1 cells and primary calvaria-derived osteoblasts, Pi increased MGP and Fra-1 expression at both the mRNA and protein levels. We also found that Pi enhanced the phosphorylation of ERK1/2. U0126 (MEK1/2 inhibitor) suppressed Pi-stimulated MGP and Fra-1 expression, indicating that ERK1/2 is required for Pi-dependent regulation of MGP and Fra-1. In addition, using in vitro DNA binding and chromatin immunoprecipitation assays, we showed that Fra-1 interacts with the MGP promoter in response to Pi in MC3T3-E1 cells. Finally, we found that in fra-1 knockdown MC3T3-E1 osteoblasts, the level of MGP expression is no more significantly upregulated by Pi. We further showed that primary osteoblasts from Fra-1-deficient mice failed to exhibit a Pi-dependent stimulation of MGP expression. These data show, for the first time, that Pi regulates MGP expression in osteoblasts through the ERK1/2-Fra-1 pathway.

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Whereas increasing evidence suggests that inorganic phosphate (Pi) may act as a signaling molecule in mineralization-competent cells, its mechanisms of action remain largely unknown. The aims of the present work were to determine whether Pi regulates expression of matrix Gla protein (MGP), a mineralization inhibitor, in growth plate chondrocytes and to identify the involved signaling pathways. Chondrogenic ATDC5 cells and primary growth plate chondrocytes were used. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting, respectively. The activation and role of MAPKs were, respectively, determined by Western blotting and the use of specific inhibitors. Immunohistological detection of ERK1/2 was performed in rib organ cultures from newborn mice. The results indicate that Pi markedly stimulates expression of MGP in ATDC5 cells and primary growth plate chondrocytes. Investigation of the involved intracellular signaling pathways reveals that Pi activates ERK1/2 in a cell-specific manner, because the stimulation was observed in ATDC5 and primary chondrocytes, MC3T3-E1 osteoblasts, and ST2 stromal cells, but not in L929 fibroblasts or C2C12 myogenic cells. Accordingly, immunohistological detection of ERK1/2 phosphorylation in rib growth plates revealed a marked signal in chondrocytes. Finally, a specific ERK1/2 inhibitor, UO126, blocks Pi-stimulated MGP expression in ATDC5 cells, indicating that ERK1/2 mediates, mainly, the effects of Pi. These data demonstrate, for the first time, that Pi regulates MGP expression in growth plate chondrocytes, thereby suggesting a key role for Pi and ERK1/2 in the regulation of bone formation.

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Physico-chemical-mechanical and in vitro biological properties of calcium phosphate cements with doped amorphous calcium phosphates.. Biomaterials, Elsevier, 2007, 28 (6)

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