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There is strong clinical evidence that implicates tenofovir in the loss of bone mineral density during treatment of human immunodeficiency virus infection. In this study, we sought to test the hypothesis that tenofovir treatment of osteoblasts causes changes in the gene expression profile that would impact osteoblast function during bone formation. Primary osteoblasts were isolated and then treated with the tenofovir prodrug, tenofovir disoproxil fumarate (TDF). Total RNA from TDF-treated and untreated osteoblasts were extracted and used for microarray analysis to assess TDF-associated changes in the gene expression profile. Strikingly, the changes in gene expression profiles involved in cell signaling, cell cycle and amino acid metabolism, which would likely impact osteoblast function in bone formation. Our findings demonstrate for the first time that tenofovir treatment of primary osteoblasts results in gene expression changes that implicate loss of osteoblast function in tenofovir-associated bone mineral density loss., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Inhibition of osteoblast-mediated mineralization is one of the major catabolic effects of parathyroid hormone (PTH) on bone. Previously, we showed that PTH induces matrix gamma-carboxyglutamic acid (Gla) protein (MGP) expression and established that this induction is critical for PTH-mediated inhibition of osteoblast mineralization. In the present study, we focus on the mechanism through which PTH regulates MGP expression in osteoblastic MC3T3-E1 cells. Following transient transfection of these cells with a -748 bp murine MGP promoter-luciferase construct (pMGP-luc), PTH (10 (-7) M) induced promoter activity in a time-dependent manner with a maximal four- to six fold induction seen 6 h after PTH treatment. Both H-89 (PKA inhibitor) and U0126 (MEK inhibitor), suppressed PTH induction of MGP promoter activity as well as the MGP mRNA level. In addition, forskolin (PKA activator) stimulated MGP promoter activity and mRNA levels confirming that PKA is one of the signaling molecules required for regulation of MGP by PTH. Co-transfection of MC3T3-E1 cells with pMGP-luc and MEK(SP), a plasmid encoding the constitutively active form of MEK, led to a dose-dependent increase in MGP promoter activity. Both MGP promoter activity and MGP mRNA level were not affected by the protein kinase C (PKC) inhibitor, GF109203X. However, phorbol 12-myristate 13-acetate (PMA), a selective PKC activator induced MGP mRNA expression through activation of extracellular signal-regulated kinase (ERK). Taken together, these results indicate that PTH regulates MGP via both PKA- and ERK-dependent pathways.

Bone morphogenetic proteins (BMPs) are potent inducers of osteoblast differentiation. The accessibility of BMP ligands for binding to their receptors is regulated by secreted proteins Twisted gastrulation (Tsg) and Chordin (Chd). Tsg antagonizes BMP signaling by forming ternary complexes with Chd and BMPs, thereby preventing BMPs from binding to their receptors. In addition to the anti-BMP function, Tsg also has pro-BMP activity, partly mediated by cleavage and degradation of Chd, which releases BMPs from ternary complexes. The roles of Tsg and Chd in osteoblast differentiation are not known. Therefore, in the present study, we investigated the effect of exogenous Tsg and Chd on osteoblast differentiation and mineralization using a well-characterized subclone of MC3T3-E1 osteoblast-like cells. Our results show that Tsg and Chd are expressed in MC3T3-E1 osteoblast-like cells. While Tsg mRNA levels decrease during osteoblast differentiation, Chd levels are found to increase. Tsg and Chd proteins accumulate in the cell culture media as the osteoblasts differentiate. Exogenous Tsg and Chd inhibit osteoblast differentiation and mineralization. Osteocalcin (OCN) mRNA levels decrease following both Tsg and Chd treatment. Tsg and Chd also inhibit alkaline phosphatase (ALP) activity in a dose-dependent manner. To provide insight into the mechanism of Tsg and Chd action, we investigated the effect of Tsg and Chd on BMP activity by determining phosphorylated Smad1 (pSmad1) levels. We show that both Tsg and Chd can independently and in combination reduce pSmad1 levels in MC3T3-E1 cells treated with BMP4. Further, BMP2 partially reverses the inhibitory effect of Tsg and Chd on ALP activity. Taken together, these results suggest that Tsg and Chd are involved in osteoblast differentiation and mineralization by regulating BMP signaling.

Parathyroid hormone (PTH) exerts biphasic effects on bone, dependent on the frequency and dose of administration. The catabolic actions of PTH on bone have been associated with continuous treatment, an increase in osteoblast-mediated resorption of bone via osteoclast activation, and inhibition of osteoblast activity and mineralization. Downregulation of differentiation markers and inhibition of mineralization by PTH have been reported in primary calvarial explants and osteoblast cell lines. Using MC3T3-E1 osteoblast-like cells, we have shown that matrix Gla protein (MGP) can be induced by PTH, and that this induction may explain the PTH-mediated inhibition of osteoblast biomineralization. MGP is a known inhibitor of mineralization, and mice deficient in Mgp show severe vascular calcification and premature bone mineralization. This review discusses the role of MGP in mineralization, comparing bone and vascular mineralization. In addition to MGP, the regulation and possible role of osteopontin, another known regulator of osteoblast mineralization, in PTH-mediated regulation of bone and vascular mineralization is discussed., (2005 S. Karger AG, Basel)