Purpose: Bisphosphonates are currently the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor-associated osteolysis and hypercalcemia. These compounds have high affinity for calcium ions and therefore target bone mineral, where they are internalized by bone-resorbing osteoclasts and inhibit osteoclast function. Experimental Design: This article reviews the pharmacology of bisphosphonates and the relationship between chemical structure and antiresorptive potency. We also describe new insights into their intracellular molecular mechanisms of action, methods for assessing the effects of bisphosphonates on protein prenylation, and their potential as direct antitumor agents. Results: Nitrogen-containing bisphosphonates act intracellularly by inhibiting farnesyl diphosphate synthase, an enzyme of the mevalonate pathway, thereby preventing prenylation of small GTPase signaling proteins required for normal cellular function. Inhibition of farnesyl diphosphate synthase also seems to account for their antitumor effects observed in vitro and for the activation of g,y T cells, a feature of the acute-phase response to bisphosphonate treatment in humans. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that seems to involve primarily the formation of cytotoxic metabolites in osteoclasts. Conclusions: Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect osteoclasts in vivo but could also have direct effects on other cell types, such as tumor cells. After >30 years of clinical use, their molecular mechanisms of action on osteoclasts are finally becoming clear but their exact antitumor properties remain to be clarified.Bisphosphonates remain the most widely used and effective antiresorptive agents for the treatment of diseases in which there is an increase in the number or activity of osteoclasts, including tumor-associated osteolysis and hypercalcemia (1). This brief review summarizes our current understanding of the molecular mechanisms of action of bisphosphonates on osteoclasts and their potential to affect other cell types, such as tumor cells, via the same molecular mechanisms. General Properties of BisphosphonatesBisphosphonates are synthetic, nonhydrolyzable analogues of PP i (Fig. 1). The P-C-P structure of bisphosphonates imparts the ability to bind divalent metal ions, such as Ca 2+ (2). For this reason, bisphosphonates are rapidly cleared from the circulation (3, 4) and bind to bone mineral surfaces in vivo at sites of active bone remodeling, particularly areas undergoing osteoclastic resorption (5). The targeting of bisphosphonates to bone, localized release during osteoclastic bone resorption, and efficient uptake into osteoclasts by endocytosis explains why bisphosphonates seem to have a highly selective effect on osteoclasts (2). However, this does not exclude the possibility that small amounts of these drugs are internaliz...
SummaryNitrogen-containing bisphosphonates indirectly activate Vc9Vd2 T cells through inhibition of farnesyl pyrophosphate synthase and intracellular accumulation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but the cells responsible for Vc9Vd2 T cell activation through IPP/DMAPP accumulation are unknown. Treatment of human peripheral blood mononuclear cells (PBMCs) with a pharmacologically relevant concentration of zoledronic acid induced accumulation of IPP/DMAPP selectively in monocytes, which correlated with efficient drug uptake by these cells. Furthermore, zoledronic acid-pulsed monocytes triggered activation of cd T cells in a cell contact-dependent manner. These observations identify monocytes as the cell type directly affected by bisphosphonates responsible for Vc9Vd2 T cell activation.
The stem cells that safeguard synovial joints in adulthood are undefined. Studies on mesenchymal stromal/stem cells (MSCs) have mainly focused on bone marrow. Here we show that lineage tracing of Gdf5-expressing joint interzone cells identifies in adult mouse synovium an MSC population largely negative for the skeletal stem cell markers Nestin-GFP, Leptin receptor and Gremlin1. Following cartilage injury, Gdf5-lineage cells underpin synovial hyperplasia through proliferation, are recruited to a Nestin-GFPhigh perivascular population, and contribute to cartilage repair. The transcriptional co-factor Yap is upregulated after injury, and its conditional ablation in Gdf5-lineage cells prevents synovial lining hyperplasia and decreases contribution of Gdf5-lineage cells to cartilage repair. Cultured Gdf5-lineage cells exhibit progenitor activity for stable chondrocytes and are able to self-organize three-dimensionally to form a synovial lining-like layer. Finally, human synovial MSCs transduced with Bmp7 display morphogenetic properties by patterning a joint-like organ in vivo. Our findings further the understanding of the skeletal stem/progenitor cells in adult life.
Background and purpose:Increased circulating levels of L-a-lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G-protein-coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55. Experimental approach: Quantitative RT-PCR was used to measure GPR55 expression in breast cancer cell lines. Cell migration and invasion were measured using a Boyden chamber chemotaxis assay and Cultrex ® invasion assay, respectively. Cell polarization and orientation in response to the microenvironment were measured using slides containing nanometric grooves. Key results: GPR55 expression was detected in the highly metastatic MDA-MB-231 breast cancer cell line. In these cells, LPI stimulated binding of [ 35 S]GTPgS to cell membranes (pEC50 6.47 Ϯ 0.45) and significantly enhanced cell chemotaxis towards serum. MCF-7 cells expressed low levels of GPR55 and did not migrate or invade towards serum factors. When GPR55 was over-expressed in MCF-7 cells, serum induced a robust migratory and invasive response, which was further enhanced by LPI and prevented by siRNA to GPR55. The physical microenvironment has been identified as a key factor in determining breast tumour cell metastatic fate. LPI endowed MDA-MB-231 cells with the capacity to detect shallow (40 nm deep) grooved slides and induced marked cancer cell polarization on both flat and grooved surfaces. Conclusions and implications: LPI and GPR55 play a role in the modulation of migration, orientation and polarization of breast cancer cells in response to the tumour microenvironment.
Bisphosphonates are effective antiresorptive agents owing to their bone-targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non-osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein-labeled risedronate (FAM-RIS) and Alexa Fluor 647–labeled risedronate (AF647-RIS), were used to address this question. Twenty-four hours after injection into 3-month-old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM-RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14high bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14+ cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone-resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo. © 2010 American Society for Bone and Mineral Research
Bisphosphonates are widely used in the treatment of diseases involving excessive bone resorption, such as osteoporosis, cancer-associated bone disease, and Paget's disease of bone. They target to the skeleton due to their calcium-chelating properties, where they primarily act by inhibiting osteoclast-mediated bone resorption. The simple bisphosphonates, clodronate, etidronate and tiludronate, are intracellularly metabolised to cytotoxic ATP analogues, while the more potent, nitrogen-containing bisphosphonates act by inhibiting the enzyme FPP synthase, thereby preventing the prenylation of small GTPases that are necessary for the normal function and survival of osteoclasts. In recent years, these concepts have been refined, with an increased understanding of the exact mode of inhibition of FPP synthase and the consequences of inhibiting this enzyme. Recent studies further suggest that the R2 side chain, as well as determining the potency for inhibiting the target enzyme FPP synthase, also influences bone mineral binding, which may influence distribution within bone and duration of action. While bisphosphonates primarily affect the function of resorbing osteoclasts, it is becoming increasingly clear that bisphosphonates may also target the osteocyte network and prevent osteocyte apoptosis, which could contribute to their anti-fracture effects. Furthermore, increasing evidence implicates monocytes and macrophages as direct targets of bisphosphonate action, which may explain the acute phase response and the anti-tumour activity in certain animal models. Bone mineral affinity is likely to influence the extent of any such effects of these agents on non-osteoclast cells. While alternative anti-resorptive therapeutics are becoming available for clinical use, bisphosphonates currently remain the principle drugs used to treat excessive bone resorption.
IntroductionThe control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs.MethodsExpression of total YAP, its paralogue transcriptional co-activator with PDZ-binding motif (TAZ), and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis, C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally, YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry.ResultsYAP, but not TAZ, was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants, however, was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1, Id2 and Id3 in response to BMP-2. In developing mouse limbs, Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage, suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo.ConclusionsOur findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0639-9) contains supplementary material, which is available to authorized users.
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