Botfly parasitism and tourism on the endangered black howler monkey of Belize.

Tourism imposes costs and benefits on wild primates. Endangered black howler monkey (Alouatta pigra) troops with high exposure to tourism had higher levels of botfly (Alouattamyia baeri) parasitism. Edge habitat and juvenile numbers did not seem to confound the observed relationship. To improve the cost/benefit ratio of tourism, we recommend further investigation.

Bone morphogenic protein 2 directly enhances differentiation of murine osteoclast precursors.

Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF-kappaB ligand (RANKL) to enhance in vitro differentiation of osteoclast-like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow-derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p-SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p-SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL-stimulated formation of multinuclear TRAP-positive cells. The BMP antagonist noggin inhibited RANKL-mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts-like cells.

Tenofovir treatment of primary osteoblasts alters gene expression profiles: implications for bone mineral density loss.

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.

Sp proteins and Runx2 mediate regulation of matrix gla protein (MGP) expression by parathyroid hormone.

As part of its catabolic action in bone, parathyroid hormone (PTH) inhibits extracellular matrix mineralization. We previously showed that PTH dose-dependently induces matrix gla protein (MGP) expression in osteoblasts and this induction is at least partially responsible for PTH-mediated inhibition of mineralization. Recently, we identified PKA and ERK/MAPK as the key signaling pathways involved in PTH regulation of MGP expression. The goal of this study was to further characterize the mechanism by which PTH stimulates expression of MGP. Deletion analysis of the murine Mgp gene promoter identified a PTH-responsive region between -173 bp and-49 bp. Using gel-mobility shift assays we found that Sp1/Sp3, and Runx2 bind to distinct sites within this region. Mutation of either the Sp or the Runx2 site reduced MGP induction by PTH, while mutation of both sites completely abolished PTH responsiveness. Overexpression of Runx2 or Sp1 activated the Mgp reporter, while Sp3 was a dose-dependent repressor of Sp1 and PTH-induced MGP expression. Collectively, these data show that PTH regulates MGP gene transcription in osteoblasts through altered activities of Sp and Runx2 transcription factors.

Twisted gastrulation and chordin inhibit differentiation and mineralization in MC3T3-E1 osteoblast-like cells.

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.

Enhanced osteoclastogenesis causes osteopenia in twisted gastrulation-deficient mice through increased BMP signaling.

The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1-deficient (Twsg1(-/-)) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1(-/-) mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1(-/-) mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL-mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1(-/-) mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1(-/-) mice was reversed in vitro in a dose-dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.

Regulation of matrix Gla protein by parathyroid hormone in MC3T3-E1 osteoblast-like cells involves protein kinase A and extracellular signal-regulated kinase pathways.

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.

Role of matrix Gla protein in parathyroid hormone inhibition of osteoblast mineralization.

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.