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1.  Bone marrow stromal cells create a permissive microenvironment for myeloma development: a new stromal role for Wnt inhibitor Dkk1 
Cancer research  2012;72(9):2183-2189.
The rapid progression of multiple myeloma is dependent upon cellular interactions within the bone marrow microenvironment. In vitro studies suggest that bone marrow stromal cells (BMSCs) can promote myeloma growth and survival and osteolytic bone disease. However, it is not possible to recreate all cellular aspects of the bone marrow microenvironment in an in vitro system, and the contributions of BMSCs to myeloma pathogenesis in an intact, immune competent, in vivo system are unknown. To investigate this, we utilized a murine myeloma model that replicates many features of the human disease. Co-inoculation of myeloma cells and a BMSC line isolated from myeloma-permissive mice in otherwise non-permissive mice resulted in myeloma development, associated with tumor growth within bone marrow and osteolytic bone disease. In contrast, inoculation of myeloma cells alone did not result in myeloma. BMSCs inoculated alone induced osteoblast suppression, associated with an increase in serum concentrations of the Wnt signaling inhibitor, Dkk1. Dkk1 was highly expressed in BMSCs and in myeloma-permissive bone marrow. Knockdown of Dkk1 expression in BMSCs decreased their ability to promote myeloma and the associated bone disease in mice. Collectively, our results demonstrate novel roles of BMSCs and BMSC-derived Dkk1 in the pathogenesis of multiple myeloma in vivo.
doi:10.1158/0008-5472.CAN-11-2067
PMCID: PMC3775476  PMID: 22374979
2.  Osteoclast derived matrix metalloproteinase-7 but not matrix metalloproteinase-9 contributes to tumor induced osteolysis 
Cancer research  2009;69(16):6747-6755.
The matrix metalloproteinases, MMP-2, -3, -7, -9 and -13 are highly expressed in the tumor-bone microenvironment and of these, MMP-7 and MMP-9 were found to be localized to bone resorbing osteoclasts in human breast to bone metastases. In a bid to define the roles of host derived MMP-7 and MMP-9 in the tumor-bone microenvironment, the tibia of MMP-7 and MMP-9 null mice were injected with osteolytic luciferase tagged mammary tumor cell lines. Our data demonstrates that osteoclast derived MMP-7 significantly contributes to tumor growth and tumor induced osteolysis while osteoclast derived MMP-9 had no impact on these processes. MMP-7 is capable of processing a number of non-matrix molecules to soluble active forms that have profound effects on cell-cell communication such as RANKL, a crucial mediator of osteoclast precursor recruitment and maturation. Therefore, the ability of osteoclast derived MMP-7 to promote RANKL solubilization in the tumor-bone microenvironment was explored. Results revealed that levels of soluble RANKL were significantly lower in the MMP-7 null mice compared to wild type controls. In keeping with this observation, MMP-7 null mice had significantly fewer osteoclast numbers at the tumor-bone interface compared to the wild-type controls. In summary, we propose that the solubilization of RANKL by MMP-7 is a potential mechanism through which MMP-7 mediates mammary tumor induced osteolysis. Our studies indicate that the selective inhibition of MMP-7 in the tumor-bone microenvironment may be of benefit for the treatment of lytic breast to bone metastases.
doi:10.1158/0008-5472.CAN-08-3949
PMCID: PMC2745595  PMID: 19679556
breast to bone metastasis; osteoclasts; osteolysis; matrix metalloproteinase; MMP-7; MMP-9; receptor activator of nuclear kappa B ligand; RANKL
3.  Bone-derived IGF mediates crosstalk between bone and breast cancer cells in bony metastases 
Cancer research  2012;72(16):4238-4249.
The continuous release of bone-stored growth factors following bone resorption promotes the colonization of circulating cancer cells. However, the precise role of each of the various growth factors remains unclear. In this study, we investigated the role of bone-derived insulin-like growth factor (IGF) in the development of bone metastases in an animal model of breast cancer. We found that local stimulation of calvarial bone resorption prior to cell inoculation stimulated subsequent bone metastases to that site in vivo, while inhibition of bone resorption inhibited bone metastases. Anchorage-independent growth of cancer cells was stimulated by the culture supernatants from resorbed bones, which contained elevated levels of IGF type I (IGF-1). This stimulation was blocked by IGF-1 receptor (IGF1R) neutralizing antibody, but not antibody targeting other bone-stored growth factors including TGFβ, fibroblast growth factors, and platelet derived growth factors. While recombinant human IGF-I caused IGFIR tyrosine autophosphorylation, followed by activation of Akt and NF-κB in cancer cells, dominant-negative inhibition of IGFIR, Akt, or NF-κB significantly reduced bone metastases with increased apoptosis and decreased mitosis in metastatic cells. Together, our findings suggest that bone-derived IGF-I bridges the crosstalk between bone and metastasized cancer cells via activation of the IGFIR/Akt/NF-κB pathway. Disruption of this pathway therefore may represent a promising therapeutic intervention for bone metastasis.
doi:10.1158/0008-5472.CAN-11-3061
PMCID: PMC3438359  PMID: 22738911
IGF receptor signaling; Akt; NF-κB; apoptosis; mitosis
4.  6-Thioguanine Inhibition of Parathyroid Hormone-related Protein Expression Is Mediated by GLI2 
Anticancer research  2011;31(9):2705-2712.
Background
Breast cancer cells frequently metastasize to bone, where they up-regulate their expression of the transcription factor GLI2 and the downstream osteolytic factor parathyroid hormone-related protein (PTHrP). The guanosine nucleotide 6-thioguanine (6-TG) inhibits PTHrP expression and blocks osteolytic bone destruction in mice inoculated with bone metastatic cells; however, the mechanism by which 6-TG inhibits PTHrP remains unclear. We hypothesized that 6-TG inhibition of PTHrP is mediated through GLI2 signaling.
Materials and Methods
Human MDA-MB-231 breast cancer cells and RWGT2 squamous-cell lung carcinoma cells were treated with 100 μM 6-TG and examined for GLI2 mRNA expression and stability by Q-PCR, promoter activity by luciferase assay, and protein expression by Western blot.
Results
6-TG significantly blocked GLI2 mRNA and protein expression, but did not affect stability. Additionally, 6-TG directly inhibited GLI2 promoter activity, and when cells were transfected with constitutively expressed GLI2, the inhibitory effect of 6-TG on PTHrP expression was abolished.
Conclusion
Taken together, these data indicate that 6-TG regulates PTHrP in part through GLI2 transcription, and therefore the clinical use of 6-TG or other guanosine nucleotides may be a viable therapeutic option in tumor types expressing elevated levels of GLI proteins.
PMCID: PMC3430074  PMID: 21868511
GLI2; PTHrP; osteolysis; breast cancer; 6-thioguanine
5.  Surface plasmon resonance (SPR) confirms that MEPE binds to PHEX via the MEPE–ASARM motif: a model for impaired mineralization in X-linked rickets (HYP) 
Bone  2004;36(1):33-46.
Matrix Extracellular Phospho-glycoprotEin (MEPE) and proteases are elevated and PHEX is defective in HYP. PHEX prevents proteolysis of MEPE and release of a protease-resistant MEPE–ASARM peptide, an inhibitor of mineralization (minhibin). Thus, in HYP, mutated PHEX may contribute to increased ASARM peptide release. Moreover, binding of MEPE by PHEX may regulate this process in normal subjects. The nature of the PHEX–MEPE nonproteolytic interaction(s) (direct or indirect) is/are unknown. Our aims were to determine (1) whether PHEX binds specifically to MEPE, (2) whether the binding involves the ASARM motif region, and (3) whether free ASARM peptide affects mineralization in vivo in mice. Protein interactions between MEPE and recombinant soluble PHEX (secPHEX) were measured using surface plasmon resonance (SPR). Briefly, secPHEX, MEPE, and control protein (IgG) were immobilized on a Biacore CM5 sensor chip, and SPR experiments were performed on a Biacore 3000 high-performance research system. Pure secPHEX was then injected at different concentrations, and interactions with immobilized proteins were measured. To determine MEPE sequences interacting with secPHEX, the inhibitory effects of MEPE–ASARM peptides (phosphorylated and nonphosphorylated), control peptides, and MEPE midregion RGD peptides on secPHEX binding to chip-immobilized MEPE were measured. ASARM peptide and etidronate-mediated mineralization inhibition in vivo and in vitro were determined by quenched calcein fluorescence in hind limbs and calvariae in mice and by histological Sanderson stain. A specific, dose-dependent and Zn-dependent protein interaction between secPHEX and immobilized MEPE occurs (EC50 of 553 nM). Synthetic MEPE PO4-ASARM peptide inhibits the PHEX–MEPE interaction (KDapp = 15 uM and Bmax/inhib = 68%). In contrast, control and MEPE–RGD peptides had no effect. Subcutaneous administration of ASARM peptide resulted in marked quenching of fluorescence in calvariae and hind limbs relative to vehicle controls indicating impaired mineralization. Similar results were obtained with etidronate. Sanderson-stained calvariae also indicated a marked increase in unmineralized osteoid with ASARM peptide and etidronate groups. We conclude that PHEX and MEPE form a nonproteolytic protein interaction via the MEPE carboxy-terminal ASARM motif, and the ASARM peptide inhibits mineralization in vivo. The binding of MEPE and ASARM peptide by PHEX may explain why loss of functional osteoblast-expressed PHEX results in defective mineralization in HYP.
doi:10.1016/j.bone.2004.09.015
PMCID: PMC3361744  PMID: 15664000
PHEX; MEPE; Rickets; Osteomalacia; Mineralization
6.  TGF-β promotion of Gli2-induced expression of parathyroid hormone-related protein, an important osteolytic factor in bone metastasis, is independent of canonical Hedgehog signaling 
Cancer research  2010;71(3):822-831.
Breast cancer frequently metastasizes to bone, where tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which up regulates expression of the Hedgehog (Hh) signaling molecule Gli2 which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 over expression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-Repressor gene (Gli2-Rep) reduced endogenous and TGF-β-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was down regulated in cells, whereas enforced over expression ofGli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression, and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.
doi:10.1158/0008-5472.CAN-10-2993
PMCID: PMC3077118  PMID: 21189326
Gli; PTHrP; Osteolysis; Breast cancer; Hedgehog; Cyclopamine; Bone Metastasis
7.  Longitudinal live animal microCT allows for quantitative analysis of tumor-induced bone destruction 
Bone  2010;48(1):141-151.
The majority of breast cancer and prostate cancer patients with metastatic disease will go on to develop bone metastases, which contribute largely to patient morbidity and mortality. Numerous small animal models of cancer metastasis to bone have been developed in order to study tumor-induced bone destruction, but the advancement of imaging modalities utilized for these models has lagged significantly behind clinical imaging. Therefore, there is a significant need for improvements to live small animal imaging, particularly when obtaining high resolution images for longitudinal quantitative analyses. Recently, live animal micro-Computed Tomography (μCT) has gained popularity due to the ability to obtain high resolution, 3-dimensional images. However, the utility of μCT in bone metastasis models has been limited to end-point analyses due to off-target radiation effects on tumor cells. We hypothesized that live animal in vivo μCT can be utilized to perform reproducible and quantitative longitudinal analyses of bone volume in tumor bearing mice, particularly in a drug treatment model of breast cancer metastasis to bone. To test this hypothesis we utilized the MDA-MB-231 osteolytic breast cancer model in which the tumor cells are inoculated directly into the tibia of athymic nude mice and imaged mice weekly by Faxitron (radiography), Imtek μCT (in vivo), and Maestro (GFP-imaging). Ex-vivo μCT and histology were performed at end-point for validation. After establishing a high resolution scanning protocol for the Imtek CT, we determined whether clear, measurable differences in bone volume were detectable in mice undergoing bisphosphonate drug treatments. We found that in vivo μCT can be used to obtain quantifiable and longitudinal images of the progression of bone destruction over time without altering tumor cell growth. Additionally, we found that we could detect lesions as early as week one and that this approach could be used to monitor the effect of drug treatment on bone. Taken together, these data indicate in vivo μCT is an effective and reproducible method for longitudinal monitoring of tumor-associated bone destruction in mouse models of tumor-induced bone disease.
doi:10.1016/j.bone.2010.05.033
PMCID: PMC2974944  PMID: 20685406
microCT; faxitron; radiography; imaging; osteolysis; bone destruction
8.  Gr-1+CD11b+ Myeloid-Derived Suppressor Cells: Formidable Partners in Tumor Metastasis 
Journal of Bone and Mineral Research  2010;25(8):1701-1706.
The growth and metastasis of solid tumors not only depends on their ability to escape from immune surveillance but also hinges on their ability to invade the vasculature system as well as to induce the formation of new blood vessels. Gr-1+CD11b+ myeloid-derived suppressor cells (MDSCs), overproduced in tumor-bearing hosts, contribute significantly to all these aspects. They also have a potential role in the osteolysis associated with bone metastases. They are formidable partners in tumor metastasis. © 2010 American Society for Bone and Mineral Research.
doi:10.1002/jbmr.154
PMCID: PMC3153347  PMID: 20572008
myeloid-derived suppressor cells; GR-1; CD11B; myeloma; metastasis; osteoclasts
9.  Local Low-Dose Lovastatin Delivery Improves the Bone-Healing Defect Caused by Nf1 Loss of Function in Osteoblasts 
Journal of Bone and Mineral Research  2010;25(7):1658-1667.
Postfracture tibial nonunion (pseudoarthrosis) leads to lifelong disability in patients with neurofibromatosis type I (NF1), a disorder caused by mutations in the NF1 gene. To determine the contribution of NF1 in bone healing, we assessed bone healing in the conditional mouse model lacking Nf1 specifically in osteoblasts. A closed distal tibia fracture protocol and a longitudinal study design were used. During the 21- to 28-day postfracture period, callus volume, as expected, decreased in wild-type but not in mice, suggesting delayed healing. At these two time points, bone volume (BV/TV) and volumetric bone mineral density (vBMD) measured by 3D micro–computed tomography were decreased in callus-bridging cortices and trabecular compartments compared with wild-type controls. Histomorphometric analyses revealed the presence of cartilaginous remnants, a high amount of osteoid, and increased osteoclast surfaces in calluses 21 days after fracture, which was accompanied by increased expression of osteopontin, Rankl, and Tgfβ. Callus strength measured by three-point bending 28 days after fracture was reduced in versus wild-type calluses. Importantly, from a clinical point of view, this defect of callus maturation and strength could be ameliorated by local delivery of low-dose lovastatin microparticles, which successfully decreased osteoid volume and cartilaginous remnant number and increased callus BV/TV and strength in mutant mice. These results thus indicate that the dysfunctions caused by loss of Nf1 in osteoblasts impair callus maturation and weaken callus mechanical properties and suggest that local delivery of low-dose lovastatin may improve bone healing in NF1 patients. © 2010 American Society for Bone and Mineral Research.
doi:10.1002/jbmr.42
PMCID: PMC3154004  PMID: 20200958
neurofibromatosis type I; NF1; fracture; healing; pseudoarthrosis; lovastatin; µCT
10.  Myeloma cells exhibit an increase in proteasome activity and an enhanced response to proteasome inhibition in the bone marrow microenvironment in vivo 
American journal of hematology  2009;84(5):268-272.
The proteasome inhibitor bortezomib has a striking clinical benefit in patients with multiple myeloma. It is unknown whether the bone marrow microenvironment directly contributes to the dramatic response of myeloma cells to proteasome inhibition in vivo. We have used the well-characterized 5TGM1 murine model of myeloma to investigate myeloma growth within bone and response to the proteasome inhibitor bortezomib in vivo. Myeloma cells freshly isolated from the bone marrow of myeloma-bearing mice were found to have an increase in proteasome activity and an enhanced response to in vitro proteasome inhibition, as compared with pre-inoculation myeloma cells. Treatment of myeloma-bearing mice with bortezomib resulted in a greater reduction in tumor burden when the myeloma cells were located within the bone marrow when compared with extra-osseous sites. Our results demonstrate that myeloma cells exhibit an increase in proteasome activity and an enhanced response to bortezomib treatment when located within the bone marrow microenvironment in vivo.
doi:10.1002/ajh.21374
PMCID: PMC2753224  PMID: 19296472
11.  Inhibition of Microtubule Assembly in Osteoblasts Stimulates Bone Morphogenetic Protein 2 Expression and Bone Formation through Transcription Factor Gli2▿  
Molecular and Cellular Biology  2008;29(5):1291-1305.
Bone morphogenetic protein 2 (BMP-2) is essential for postnatal bone formation and fracture repair. By screening chemical libraries for BMP-2 mimics using a cell-based assay, we identified inhibitors of microtubule assembly as stimulators of BMP-2 transcription. These microtubule inhibitors increased osteoblast differentiation in vitro, stimulated periosteal bone formation when injected locally over murine calvaria, and enhanced trabecular bone formation when administered systemically in vivo. To explore molecular mechanisms mediating these responses, we examined effects of microtubule inhibitors on the hedgehog (Hh) pathway, since this pathway is known to regulate BMP-2 transcription in osteoblasts and microtubules have been shown to be involved in Hh signaling in Drosophila. Here we show that in osteoblasts, inhibition of microtubule assembly increased cytoplasmic levels and transcriptional activity of Gli2, a transcriptional mediator of Hh signaling that we have previously shown to enhance BMP-2 expression in osteoblasts (M. Zhao et al., Mol. Cell. Biol. 26:6197-6208, 2006). Microtubule inhibition blocked β-TrCP-mediated proteasomal processing of Gli2 in osteoblasts. In summary, inhibition of microtubule assembly enhances BMP-2 gene transcription and subsequent bone formation, in part, through inhibiting proteasomal processing of Gli2 and increasing intracellular Gli2 concentrations.
doi:10.1128/MCB.01566-08
PMCID: PMC2643819  PMID: 19103752
12.  The Pathogenesis of the Bone Disease of Multiple Myeloma 
Bone  2008;42(6):1007-1013.
Multiple myeloma is a fatal hematologic malignancy associated with clonal expansion of malignant plasma cells within the bone marrow and the development of a destructive osteolytic bone disease. The principal cellular mechanisms involved in the development of myeloma bone disease are an increase in osteoclastic bone resorption, and a reduction in bone formation. Myeloma cells are found in close association with sites of active bone resorption, and the interactions between myeloma cells, and other cells within the specialized bone marrow microenvironment are essential, both for tumor growth and the development of myeloma bone disease. This review discusses the many different factors which have been implicated in myeloma bone disease, including the evidence for their role in myeloma and subsequent therapeutic implications.
doi:10.1016/j.bone.2008.01.027
PMCID: PMC2474770  PMID: 18406675
13.  Smurf1 Inhibits Osteoblast Differentiation and Bone Formation in Vitro and in Vivo* 
The Journal of biological chemistry  2003;279(13):12854-12859.
Bone morphogenetic proteins (BMPs) are required for normal postnatal bone formation and osteoblast differentiation. There is evidence from recent studies that BMP signaling in osteoblasts is controlled by an ubiquitin-proteasome regulatory mechanism involving a cascade of enzymatic reactions. The specificity of protein ubiquitination is determined by E3 ubiquitin ligases, which play a crucial role in defining substrate specificity and subsequent protein degradation by 26S proteasomes. We have examined the role of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1), a member of the Hect domain family of E3 ubiquitin ligases in osteoblast function. Smurf1 has been found to interact with BMP-activated Smad1 and -5 and to mediate degradation of these Smad proteins. Recently we have found that Smurf1 mediates the protein degradation of the osteoblast-specific transcription factor Runx2/Cbfa1. To determine the role of Smurf1 in osteoblast differentiation, in the present studies we transfected a Smurf1 expression plasmid into 2T3 osteoblast precursor cells and found that Smurf1 overexpression inhibits BMP signaling and osteoblast differentiation. To further investigate the role of Smurf1 in bone formation in vivo, we generated transgenic mice in which expression of the epitope-tagged Smurf1 transgene was targeted to osteoblasts using the murine 2.3-kb osteoblast-specific type I collagen promoter. In these transgenic mice, bone formation was significantly reduced during postnatal life. Our results demonstrate for the first time that Smurf1 plays a specific role in osteoblast differentiation and bone formation in vivo.
doi:10.1074/jbc.M313294200
PMCID: PMC2649829  PMID: 14701828
14.  Caspase-2 Deficiency Enhances Aging-Related Traits in Mice 
Alteration of apoptotic activity has been observed in a number of tissues in aging mammals, but it remains unclear whether and/or how apoptosis may affect aging. Caspase-2 is a member of the cysteine protease family that plays a critical role in apoptosis. To understand the impact of compromised apoptosis function on mammalian aging, we conducted a comparative study on caspase-2 deficient mice and their wild-type littermates with a specific focus on the aging-related traits at advanced ages. We found that caspase-2 deficiency enhanced a number of traits commonly seen in premature aging animals. Loss of caspase-2 was associated with shortened maximum lifespan, impaired hair growth, increased bone loss, and reduced body fat content. In addition, we found that the livers of caspase-2 deficient mice had higher levels of oxidized proteins than those of age-matched wild-type mice, suggesting that caspase-2 deficiency compromised the animal's ability to clear oxidatively damaged cells. Collectively, these results suggest that caspase-2 deficiency affects aging in the mice. This study thus demonstrates for the first time that disruption of a key apoptotic gene has a significant impact on aging.
doi:10.1016/j.mad.2006.11.030
PMCID: PMC1828128  PMID: 17188333
caspase-2; maximum lifespan; bone; hair growth; fat
15.  Eph Receptors and Ephrin Signaling Pathways: A Role in Bone Homeostasis 
The maintenance of bone homeostasis is tightly controlled, and largely dependent upon cellular communication between osteoclasts and osteoblasts, and the coupling of bone resorption to bone formation. This tight coupling is essential for the correct function and maintenance of the skeletal system, repairing microscopic skeletal damage and replacing aged bone. A range of pathologic diseases, including osteoporosis and cancer-induced bone disease, disrupt this coupling and cause subsequent alterations in bone homeostasis. Eph receptors and their associated ligands, ephrins, play critical roles in a number of cellular processes including immune regulation, neuronal development and cancer metastasis. Eph receptors are also expressed by cells found within the bone marrow microenvironment, including osteoclasts and osteoblasts, and there is increasing evidence to implicate this family of receptors in the control of normal and pathological bone remodeling.
PMCID: PMC2536716  PMID: 18797510
Bone remodeling; Eph receptors; ephrins; coupling; osteoblast; osteoclast
16.  The Zinc Finger Transcription Factor Gli2 Mediates Bone Morphogenetic Protein 2 Expression in Osteoblasts in Response to Hedgehog Signaling 
Molecular and Cellular Biology  2006;26(16):6197-6208.
Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. We hypothesize that Gli2 regulates BMP-2 gene transcription and thus osteoblast differentiation. In the present study, we show that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in osteoblast precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal osteoblast differentiation.
doi:10.1128/MCB.02214-05
PMCID: PMC1592805  PMID: 16880529
17.  Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation 
The Journal of Cell Biology  2002;157(6):1049-1060.
Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.
doi:10.1083/jcb.200109012
PMCID: PMC2174055  PMID: 12058020
BMP; receptor; transgenic mice; osteoblast differentiation; bone formation
18.  Guanosine nucleotides inhibit different syndromes of PTHrP excess caused by human cancers in vivo 
The Journal of Clinical Investigation  2002;110(10):1559-1572.
There are two well-described syndromes caused by tumor production of parathyroid hormone-related peptide (PTHrP), namely osteolytic bone disease associated with breast cancer and humoral hypercalcemia of malignancy (HHM) that occurs with or without bone metastasis. Both syndromes have been shown experimentally to be inhibited by neutralizing antibodies to PTHrP. In a search for small-molecule inhibitors of PTHrP production or effects, we have identified guanine-nucleotide analogs as compounds that inhibit PTHrP expression by human tumor cells associated with these syndromes. We show in nude athymic murine models that these compounds reduce PTHrP-mediated osteolytic lesions associated with metastatic human breast-cancer cells as well as the degree of hypercalcemia caused by excessive PTHrP production by a squamous-cell carcinoma of the lung. These results suggest that the PTHrP gene promoter may be a suitable target for treating the skeletal effects of malignancy.
doi:10.1172/JCI11936
PMCID: PMC151806  PMID: 12438453
19.  Decreased C-Src Expression Enhances Osteoblast Differentiation and Bone Formation 
The Journal of Cell Biology  2000;151(2):311-320.
c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src−/− relative to Src+/+ mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by ∼60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src−/− osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.
PMCID: PMC2192638  PMID: 11038178
osteopetrosis; Src; osteoblast; differentiation; bone formation
20.  TGF-β signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development 
Journal of Clinical Investigation  1999;103(2):197-206.
Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-β (TGF-β), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-β inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-β responses, any role of TGF-β in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-β promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant–negative mutant (TβRIIΔcyt) of the TGF-β type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-β. In a murine model of bone metastases, expression of TβRIIΔcyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant–negative signaling blockade by expression of a constitutively active TGF-β type I receptor in the breast cancer cells increased tumor production of parathyroid hormone–related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant–negative TβRIIΔcyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-β in the development of breast cancer metastasis to bone, via the TGF-β receptor–mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.
PMCID: PMC407876  PMID: 9916131
21.  Cadherin-6 Mediates the Heterotypic Interactions between the Hemopoietic Osteoclast Cell Lineage and Stromal Cells in a Murine Model of Osteoclast Differentiation  
The Journal of Cell Biology  1998;141(6):1467-1476.
Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell–cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell–cell contact caused evident morphological changes accompanied with tight cell–cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow–derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.
PMCID: PMC2132794  PMID: 9628901
22.  Collagen and Collagen-derived Fragments Are Chemotactic for Tumor Cells 
Journal of Clinical Investigation  1981;68(4):1102-1105.
Organs that are rich in collagen such as liver, lungs, and bone are frequently sites of tumor cell metastasis. In this study, we have found that cultured tumor cells of human and rat origin migrated unidirectionally in response to collagen in vitro. Synthetic di- and tri-peptides that contained amino acid sequences found frequently in the collagen helix caused similar effects. These results are consistent with the hypothesis that collagen or collagen fragments released during connective tissue remodeling may be important in tumor cell metastasis.
PMCID: PMC370899  PMID: 7287902
23.  Observations on the Mechanism of Bone Resorption Induced by Multiple Myeloma Marrow Culture Fluids and Partially Purified Osteoclast-activating Factor 
Journal of Clinical Investigation  1981;67(5):1472-1481.
Supernatant fluids from the cultures of bone marrow cells from 10 of 12 patients with multiple myeloma (MM) caused bone resorption in organ cultures of fetal rat calvaria. In four patients, the marrow cells were cultured with and without indomethacin (1 μM). The supernatant fluids from indomethacintreated marrow cultures caused significantly less bone resorption than supernatant fluids of cell cultures without indomethacin. This inhibition of release of bone resorbing factor(s) by myeloma cultures is similar to the previously observed indomethacin-induced inhibition of osteoclast-activating factor (OAF) production by activated human leukocytes. None of the MM supernatants had any effect on cyclic (c)AMP accumulation in resorbing bone in vitro.
Four separate preparations of partially purified OAF obtained from phytohemagglutinin-stimulated peripheral human leukocytes were tested for their ability (a) to cause bone resorption in organ cultures of fetal rat and neonatal mouse calvaria and (b) to cause accumulation of cAMP in rat and mouse skeletal tissue in vitro. Those dilutions of OAF that caused bone resorption had no effect on accumulation of cAMP in rat or mouse calvaria incubated in vitro. In addition, no stimulation of adenylate cyclase activity in membranes prepared from fetal rat calvaria could be found. Bone cell populations isolated by sequential collagenase digestion of fetal rat calvaria also showed no cAMP response to these dilutions of OAF. Parathyroid hormone caused a clear response in all three systems. Furthermore, no cAMP response to OAF was observed in calvaria in the presence of cholera toxin (1 μg/ml) and isobutyl-methylxanthine (0.3 mM).
These observations demonstrate that (a) supernatant fluids from MM marrow cultures stimulate bone resorption but do not increase cAMP accumulation in vitro; (b) indomethacin interferes with the release of bone resorbing factors by MM bone marrow cultures suggesting that this process requires prostaglandins; and (c) Sephadex G100 or G75 purified OAF does not stimulate adenylate cyclase or increase cAMP accumulation at equivalent bone resorbing concentrations in rat and mouse skeletal tissue.
The resorptive action of MM culture fluids is similar to that of partially purified OAF from activated cultured leukocytes, but different from those of other bone resorbing factors, parathyroid hormone and prostaglandin E2, which stimulate cAMP production in skeletal tissue.
PMCID: PMC370715  PMID: 6262378
24.  Big and Little Forms of Osteoclast Activating Factor 
Journal of Clinical Investigation  1977;60(1):122-128.
We have further characterized osteoclast activating factor (OAF) using a bioassay for bone resorption which utilizes the release of previously incorporated 45Ca from fetal rat long bones in organ culture. When supernatant media from activated leukocyte cultures were concentrated on Amicon PM10 membranes (assigned molecular weight cutoff 10,000 daltons) and chromatographed on Sephadex G-50 columns, the bone-resorbing activity eluted between the molecular weight markers chymotrypsinogen (25,000 daltons) and cytochrome c (12,500 daltons). This peak of biological activity has been called big OAF. When filtrates from the PM10 membranes were concentrated on Amicon UM2 membranes (assigned molecular weight cutoff 1,000 daltons) and chromatographed on Sephadex G-50 columns, some of the biological activity eluted between the molecular weight markers chymotrypsinogen and cytochrome c (big OAF), but there was a separate peak of biological activity which eluted with [3H]proline (140 daltons). This second peak has been called little OAF. Little OAF was eluted from Bio-Gel P6 columns between the molecular weight markers calcitonin (approximately 3,500 daltons) and vitamin B12 (1,330 daltons), but was retained by Spectrapor dialysis tubing (nominal molecular weight cutoff 3,500 daltons). Big OAF was converted to little OAF by equilibration in 1 M NaCl or 2 M urea. Little OAF was self-associated back to big OAF by equilibration in buffers of low ionic strength (Tris-HCl 10-50 mM). Little OAF was extracted into the organic phase in ethyl acetate after acidification of the sample to pH 3.5. The biological activity remained in the aqueous phase after ethyl acetate extraction at pH 7.5-8.4. Little OAF has been purified more than 6,000-fold compared with the original material so that bone-resorbing activity is maximal in a sample with a protein concentration of 80 ng/ml.
PMCID: PMC372350  PMID: 874077
25.  Partial Purification of Osteoclast-Activating Factor from Phytohemagglutinin-Stimulated Human Leukocytes 
Journal of Clinical Investigation  1974;53(5):1473-1480.
Osteoclast-activating factor (OAF) is a soluble mediator found in supernates of human peripheral leukocytes which have been cultured with antigens or phytomitogens. OAF is a potent stimulator of osteoclastic resorption of fetal bone in organ culture. The present studies were designed to characterize OAF chemically. Bone resorbing activity from supernates of leukocytes cultured without added plasma was not lost on dialysis using a membrane with a molecular weight cutoff of 3,500, but was lost when heated to 60°C for 30 min. The activity was lost after treatment with trypsin or pronase but not after treatment with ribonuclease or neuraminidase. Papain, which inactivated parathyroid hormone at a concentration of 25 μg/ml, did not inactivate OAF at 250 μg/ml. OAF did not react with an antibody to bovine parathyroid hormone which cross-reacts with human parathyroid hormone. OAF was also distinguished from active metabolites of vitamin D and from prostaglandin by extraction procedures and immunoassay for prostaglandin E2.
When the medium from activated leukocytes cultured with autologous plasma was fractionated by gel filtration on Sephadex, bone resorbing activity eluated both with plasma proteins and in lower molecular weight fractions. However, when medium from leukocytes cultured without added plasma was chromatographed, all the OAF activity was eluted in a sharp low molecular weight peak located between chymotrypsinogen (25,000 molecular weight) and ribonuclease A (13,700 molecular weight). This peak contained about 4% of the total protein originally present in the supernate. Its activity was destroyed by overnight incubation at 37°C at pH 6 or 8, but not at pH 7.2. After incubation at 4°C, the activity was lost at pH 3 or 10, but not at pH 4-9.
The active fraction from Sephadex G-100 was therefore chromatographed at pH 7.2 on DEAE cellulose and carboxymethyl cellulose. The active material was not adsorbed; however, about sevenfold further purification was achieved by removal of contaminants. The material obtained after sequential Sephadex, DEAE and, carboxymethyl cellulose chromatography stimulated resorption of fetal rat bone in culture at concentrations of 0.75-3 μg protein/ml, indicating that this preparation of OAF was nearly as potent as bovine parathyroid hormone in this system.
PMCID: PMC302636  PMID: 4825237

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