Genetic polymorphisms of EPHX1, Gsk3beta, TNFSF8 and myeloma cell DKK-1 expression linked to bone disease in myeloma.

Bone disease in myeloma occurs as a result of complex interactions between myeloma cells and the bone marrow microenvironment. A custom-built DNA single nucleotide polymorphism (SNP) chip containing 3404 SNPs was used to test genomic DNA from myeloma patients classified by the extent of bone disease. Correlations identified with a Total Therapy 2 (TT2) (Arkansas) data set were validated with Eastern Cooperative Oncology Group (ECOG) and Southwest Oncology Group (SWOG) data sets. Univariate correlates with bone disease included: EPHX1, IGF1R, IL-4 and Gsk3beta. SNP signatures were linked to the number of bone lesions, log(2) DKK-1 myeloma cell expression levels and patient survival. Using stepwise multivariate regression analysis, the following SNPs: EPHX1 (P=0.0026); log(2) DKK-1 expression (P=0.0046); serum lactic dehydrogenase (LDH) (P=0.0074); Gsk3beta (P=0.02) and TNFSF8 (P=0.04) were linked to bone disease. This assessment of genetic polymorphisms identifies SNPs with both potential biological relevance and utility in prognostic models of myeloma bone disease.

Locally delivered lovastatin nanoparticles enhance fracture healing in rats.

Statins stimulate bone formation in vitro and in vivo and, when given in large doses or by prolonged infusions, stimulate biomechanical strength of murine long bones with healing fractures. However, administration of statins by large oral doses or prolonged infusions to a fracture site is not a feasible therapeutic approach to hasten healing of human fractures. We administered lovastatin in biodegradable polymer nanobeads of poly(lactic-co-glycolide acid) to determine if lovastatin delivered in low doses in nanoparticles of a therapeutically acceptable scaffold could increase rates of healing in a standard preclinical model of femoral fracture. We found that these nanobeads: (1) stimulated bone formation in vitro at 5 ng/mL, (2) increased rates of healing in femoral fractures when administered as a single injection into the fracture site, and (3) decreased cortical fracture gap at 4 weeks as assessed by microcomputed tomography. These preclinical results suggest that lovastatin administered in a nanobead preparation may be therapeutically useful in hastening repair of human fractures.

Transdermal application of lovastatin to rats causes profound increases in bone formation and plasma concentrations.

INTRODUCTION: Statins are drugs that inhibit HMG Co-A reductase and have been shown to enhance bone formation in vitro and in vivo in rodents. However, the statins currently used for cholesterol-lowering have been selected for their capacity to target the liver where their effects on cholesterol synthesis are mediated and they undergo first pass metabolism. When given in lipid-lowering doses, these agents do not likely reach sufficient blood concentrations to reliably cause substantial increases in bone formation in humans. Moreover, statins are inactivated by cytochrome P450 enzymes, resulting in even less peripheral distribution of the biologically active moieties beyond the liver. METHOD: To investigate whether an alternate method of administration might produce beneficial effects on bone formation, we administered lovastatin by dermal application to rats to circumvent the first-pass effects of the gut wall and liver. RESULTS: We found that the statin blood levels measured by HMG Co-A reductase activity were higher, maintained longer and less variable following transdermal application than those following oral administration. Also the increased circulating statin levels were associated with significantly enhanced biological effects on bone. After only 5 days of administration of transdermal lovastatin to rats, there was a 30-60% increase in trabecular bone volume, and 4 weeks later, we observed more than a 150% increase in bone formation rates. There was also a significant increase in serum osteocalcin, a marker of bone formation. We also found that lovastatin administered transdermally produces these profound effects at doses in the range of 1% of the oral dose, without any evidence of the hepatotoxicity or myotoxicity that can occur following oral statin administration. Several doses (0.01-5 mg kg(-1) day(-1)) and dosage schedules were examined, and collectively the data strongly suggest a powerful anabolic effect but with an unusually flat dose-response curve. CONCLUSION: These results show transdermal application of statins produces greater beneficial effects on bone formation than oral administration does.

MEPE has the properties of an osteoblastic phosphatonin and minhibin.

Matrix extracellular phosphoglycoprotein (MEPE) is expressed exclusively in osteoblasts, osteocytes and odontoblasts with markedly elevated expression found in X-linked hypophosphatemic rickets (Hyp) osteoblasts and in oncogenic hypophosphatemic osteomalacia (OHO) tumors. Because these syndromes are associated with abnormalities in mineralization and renal phosphate excretion, we examined the effects of insect-expressed full-length human-MEPE (Hu-MEPE) on serum and urinary phosphate in vivo, (33)PO(4) uptake in renal proximal tubule cultures and mineralization of osteoblast cultures. Dose-dependent hypophosphatemia and hyperphosphaturia occurred in mice following intraperitoneal (IP) administration of Hu-MEPE (up to 400 microg kg(-1) 31 h(-1)), similar to mice given the phosphaturic hormone PTH (80 microg kg(-1) 31 h(-1)). Also the fractional excretion of phosphate (FEP) was stimulated by MEPE [65.0% (P < 0.001)] and PTH groups [53.3% (P < 0.001)] relative to the vehicle group [28.7% (SEM 3.97)]. In addition, Hu-MEPE significantly inhibited (33)PO(4) uptake in primary human proximal tubule renal cells (RPTEC) and a human renal cell line (Hu-CL8) in vitro (V(max) 53.4% inhibition; K(m) 27.4 ng/ml, and V(max) 9.1% inhibition; K(m) 23.8 ng/ml, respectively). Moreover, Hu-MEPE dose dependently (50-800 ng/ml) inhibited BMP2-mediated mineralization of a murine osteoblast cell line (2T3) in vitro. Inhibition of mineralization was localized to a small (2 kDa) cathepsin B released carboxy-terminal MEPE peptide (protease-resistant) containing the acidic serine-aspartate-rich motif (ASARM peptide). We conclude that MEPE promotes renal phosphate excretion and modulates mineralization.

Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro.

We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic beta subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor-1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss.

Autoregulation of mouse BMP-2 gene transcription is directed by the proximal promoter element.

Bone morphogenetic protein-2 (BMP-2) stimulates the commitment and differentiation of precursor mesenchymal cells to mature bone. We have isolated and sequenced 2712 base pairs (bp) of the 5' flanking region of mouse BMP-2 gene. Using RNase protection assay we identified two transcription initiation sites within this 2712 bp region of the BMP-2 gene. The distal start site was mapped to -736 bp in relation to the proximal start site (+1). Recombinant BMP-2 preferentially stimulated transcription initiation from the proximal start site. To investigate the mechanism of transcription initiation from these two start sites, we identified two promoter elements upstream of the proximal and distal transcription initiation sites. Transfection of promoter-luciferase reporter constructs into cells of different organs demonstrated differential transcriptional activity of proximal and distal promoters, with highest activity in the osteoblast cell lineage. In osteoblasts, BMP-2 stimulated transcription from the proximal promoter only. Together our data provide the first evidence for the presence of two transcription initiation sites with two upstream promoter elements in mouse BMP-2 gene. Furthermore, we demonstrate for the first time that BMP-2 autoregulates its expression in osteoblasts through the proximal promoter-dependent transcriptional mechanism.

Bone morphogenetic protein-2 (BMP-2) signaling to the Col2alpha1 gene in chondroblasts requires the homeobox gene Dlx-2.

To understand the role of Dlx genes in the process of chondrogenesis, we studied the expression of Dlx-2 and Dlx-5 mRNAs in a mouse clonal chondroblast cell line, TMC23. We also examined the involvement of Dlx2 in the bone morphogenetic protein-2 (BMP-2) signaling to the type II collagen gene, Col2alpha1, in this cell line. In this report, we show that the TMC23 cells express Dlx-2 and Dlx-5 mRNAs, and the levels can be upregulated by recombinant BMP-2 at an early stage of chondroblast differentiation. Addition of rBMP-2 dramatically increased type II collagen expression at both the mRNA and the protein level. Also, rBMP-2 increased transcription of Col2alpha1, as shown by stimulation of a chondrocyte-specific Col2alpha1 enhancer. The mechanism involves Dlx-2, as the stimulatory effect of rBMP-2 on the Col2alpha enhancer was blocked by an antisense oligonucleotide against Dlx-2 mRNA. The rBMP-2 signaling to the Col2alpha1 enhancer was also blocked by a dominant-negative Smad1 expression vector. These data demonstrate that Dlx-2 is a downstream target of the BMP-2 signaling pathway in chondroblasts. Therefore, we propose a model in which rBMP-2 stimulates Dlx-2 expression, which then serves as a necessary transcription factor for Col2alpha1 gene expression through a chondrocyte-specific enhancer fragment.

The bisphosphonate ibandronate promotes apoptosis in MDA-MB-231 human breast cancer cells in bone metastases.

Bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used as a beneficial agent for the treatment of bone metastases in patients with breast cancer. It is well recognized that BP reduces osteolysis by promoting apoptosis in osteoclasts. However, recent animal and human data suggest that BPs not only reduce osteolysis associated with metastatic breast cancer, but also decrease tumor burden in bone. The mechanisms by which tumor burden is decreased following BP administration are unknown. Here we examined the effects of the BP ibandronate on MDA-231 human breast cancer cells in bone metastases in a well-characterized animal model of bone metastasis. Ibandronate, which was administered (s.c. daily; 4 microg/mouse/day) after bone metastases were established, inhibited the progression of established osteolytic bone metastases as assessed by radiographic analysis. Histological and histomorphometrical examination revealed that ibandronate reduced osteoclastic bone resorption, with increased apoptosis in osteoclasts. Furthermore, ibandronate also significantly decreased the MDA-231 tumor burden, with increased apoptosis in MDA-231 breast cancer cells in bone metastases. In contrast, ibandronate failed to inhibit MDA-231 tumor formation with no effects on apoptosis in MDA-231 breast cancer cells in the orthotopic mammary fat pads. These data suggest that the effects of ibandronate on apoptosis in MDA-231 breast cancer cells are restricted in bone in which ibandronate selectively deposits. Consistent with these in vivo results, a relatively high concentration of ibandronate (100 microM) increased caspase-3 activity and induced DNA fragmentation in MDA-231 breast cancer cells in culture. Moreover, a caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone, blocked ibandronate-induced DNA fragmentation in MDA-231 cells, suggesting an involvement of caspase-3 in ibandronate-induced apoptosis. Our results suggest that BP suppresses bone metastases through promotion of apoptosis in metastatic cancer cells as well as in osteoclasts. However, it still remains open whether BP has direct anticancer actions in vivo.

Statins and bone formation.

The main therapy needed most in the bone field is an anabolic agent for the treatment of osteoporosis. Current drugs on the market, which included bisphosphonates, calcitonin, estrogen and related compounds, vitamin D analogues trabecular microarchitecture. Therefore, it would be desirable to have a satisfactory and universally and iprifalvone, are essentially bone resorption inhibitors that mainly act to stabilize bone mass. Patients with established osteoporosis have lost more than 50% of their bone mass at critical sites in the skeleton, and more over have marked disruption of acceptable drug that would stimulate new bone formation and correct this disturbance of trabecular microarchitecture characteristic of established osteoporosis. Recently inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which controls the first step in the biosynthesis of cholesterol, have been shown to stimulate bone formation in rodents both in vitro and in vivo. The effect is associated with an increased expression of the bone morphogenetic protein-2 (BMP-2) gene in bone cells. These statins drugs are widely used agents for lowering cholesterol and reducing heart attacks, however they are also known to elicit numerous pleiotropic effects including inhibition of proliferation and migration of smooth muscle cells, inhibition of tumor growth and anti-inflammatory activity. Some of these effects have been attributed to not only to the reduction of cholesterol synthesis by inhibition of the HMG-CoA reductase enzyme but also by the concurrent reduction in downstream metabolites of the mevalonate pathway such as mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate. The findings that statins are capable of increasing bone formation and bone mass in rodents suggests a potential new action for the statins, which may be beneficial in patients with established osteoporosis where marked bone loss has occurred. Recent clinical data suggests that they may reduce the risk of fracture in patients taking these drugs. However, their precise role can only be determined by appropriate randomized clinical trials, which demonstrate their efficacy in this regard in patients.

Preclinical studies with zoledronic acid and other bisphosphonates: impact on the bone microenvironment.

The propensity for breast cancer cells to metastasize to bone and to induce osteolysis has long been recognized. Characteristics of both the tumor cells and the bone microenvironment contribute to this phenomenon. The presence of tumor in bone is associated with activation of osteoclasts, resulting in excessive bone resorption and subsequent osteolysis. Breast cancer cells and other tumor types influence osteoclastic bone resorption by increasing the number of osteoclasts and enhancing their resorptive activity. Parathyroid hormone-related peptide, in addition to its role in humorally mediated hypercalcemia, is secreted by metastatic breast cancer cells in bone in which it acts as a paracrine factor to stimulate osteoclasts. As bone matrix is broken down by activated osteoclasts, a rich supply of mitogenic factors is released, including insulin-like growth factors, bone morphogenetic proteins, and fibroblast growth factors. Transforming growth factor (TGF)-beta, one of the most abundant of the bone-derived factors, promotes increased production of parathyroid hormone-related peptide by tumor cells, establishing a "vicious cycle" leading to progressive tumor growth and bone destruction. Bisphosphonates interrupt this cycle by inhibiting osteoclasts, in part by inducing osteoclast apoptosis. In several animal models of breast cancer metastasis to bone, bisphosphonates decrease the number of new bone metastases and inhibit progression of existing lesions. A single 3 microg intravenous injection of zoledronic acid (Zometa; Novartis Pharmaceuticals Corp, East Hanover, NJ), a new highly potent bisphosphonate, prevented destruction of trabecular bone in an orthotopic mouse mammary tumor model. Tumor volume in bone was decreased by zoledronic acid in a dose-dependent manner in the same model, and tumor cell apoptosis was increased by zoledronic acid in bone metastases in the 4T1 murine model of mammary carcinoma metastasis. Zoledronic acid at a dose of 1.0 microg/d for 10 days also reduced bone lesion area in a nude mouse model with existing bone metastases. Although bisphosphonates, including zoledronic acid, are able to induce apoptosis in tumor cells in vitro, studies in animal models to date have generally not shown a reduction in nonosseous tumor. Therefore, bisphosphonate-associated tumor reduction in bone is most likely mediated by osteoclast inhibition or is related to high local concentrations of bisphosphonates in the bone compartment.

Therapeutic efficacy of a soluble receptor activator of nuclear factor kappaB-IgG Fc fusion protein in suppressing bone resorption and hypercalcemia in a model of humoral hypercalcemia of malignancy.

Receptor activator of nuclear factor kappaB (RANK) is a membrane-bound tumor necrosis factor receptor homologue that mediates signals obligatory for osteoclastogenesis as well as osteoclast activation and survival in vivo. The present study was undertaken to evaluate the efficacy of a soluble murine RANK-human immunoglobulin fusion protein (muRANK.Fc) as a bone resorption inhibitor in vitro and in vivo. The in vitro studies demonstrated the ability of muRANK.Fc to inhibit human parathyroid hormone-related protein (PTHrP)-induced resorption in fetal rat long bone cultures. Short-term administration of muRANK.Fc to normal growing mice resulted in a complete disappearance of osteoclasts from metaphyses of long bones associated with a pronounced increase in calcified trabeculae and bone radiodensity. In a model of humoral hypercalcemia of malignancy in which PTHrP secreted by s.c. xenografts of human lung cancer in nude mice induces extensive osteolysis and severe hypercalcemia, daily administration of muRANK.Fc from time of tumor implantation profoundly inhibited osteoclastic bone resorption and prevented hypercalcemia. muRANK.Fc had no effect on tumor production of PTHrP, because there was no significant difference between circulating human PTHrP levels in muRANK.Fc-treated and vehicle-treated tumor-bearing mice. Moreover, even when treatment was initiated after hypercalcemia was established, muRANK.Fc significantly attenuated further increases in blood ionized calcium. These data demonstrate the potent antiresorptive effects of muRANK.Fc in vivo as well as highlight the potential utility of disrupting RANK signaling as a novel therapeutic approach in humoral hypercalcemia of malignancy and possibly multiple myeloma and skeletal metastases associated with osteolysis.

Osteoporosis: pathophysiology and non-pharmacological management.

Osteoporosis is the disease of progressive bone loss that is most often associated with ageing and the post-menopausal state in women. All people, men and women, lose bone mass with advancing age, but in some the loss is so great that the skeleton is unable to maintain optimal structural integrity and the result is susceptibility to fractures, particularly of the hip and spine. The condition is increasing dramatically in prevalence as the numbers of elderly in the population increase. In this chapter, the epidemiology, clinical features and current modes of non-pharmacological management of osteoporosis are reviewed, with discussion of the potential of nutraceuticals and functional foods to influence the course of the disease.

Decreased c-Src expression enhances osteoblast differentiation and bone formation.

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 approximately 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.

Optimizing human demineralized bone matrix for clinical application.

The use of human demineralized bone matrix (DBM) powder in periodontal and orthopedic applications is limited by the variability in the osteoinductive or osteoconductive properties of the material. The goal of the present study was to establish simple in vitro and in vivo assays of DBM that would allow us to screen different lots of the material prior to testing in more rigorous animal models. The results demonstrate a wide variability in the performance of individual lots of DBM powder obtained from a single tissue bank. The studies also demonstrate that relatively simple screening can be used to establish the quality of the different lots, and that performance and ease of handling can be improved by using relatively small particle sizes delivered in a fibrin sealant matrix.

Pathogenesis of osteoporosis and challenges for drug delivery.

Bone remodeling is under the control of local factors produced in the bone microenvironment. Many of the mechanisms responsible for coordination of activity between osteoclasts and osteoblasts, and how they are dysregulated in diseases of bone loss such as osteoporosis, are now being clarified. This leads to the possibility that bone remodeling can be manipulated by pharmacological means to reverse the changes caused by common bone diseases. However, many of the potential drugs suffer from non-selectivity in their effects. Targeting of drugs to active skeletal sites is of major interest to the pharmaceutical industry, and there are interesting and exciting possibilities which could lead to more specific therapies for the common diseases of bone loss. These will be reviewed in this chapter.

Cell-cell contact between marrow stromal cells and myeloma cells via VCAM-1 and alpha(4)beta(1)-integrin enhances production of osteoclast-stimulating activity.

Myeloma is a unique hematologic malignancy that exclusively homes in the bone marrow and induces massive osteoclastic bone destruction presumably by producing cytokines that promote the differentiation of the hematopoietic progenitors to osteoclasts (osteoclastogenesis). It is recognized that neighboring bone marrow stromal cells influence the expression of the malignant phenotype in myeloma cells. This study examined the role of the interactions between myeloma cells and neighboring stromal cells in the production of osteoclastogenic factors to elucidate the mechanism underlying extensive osteoclastic bone destruction. A murine myeloma cell line 5TGM1, which causes severe osteolysis, expresses alpha(4)beta(1)-integrin and tightly adheres to the mouse marrow stromal cell line ST2, which expresses the vascular cell adhesion molecule-1 (VCAM-1), a ligand for alpha(4)beta(1)-integrin. Co-cultures of 5TGM1 with primary bone marrow cells generated tartrate-resistant acid phosphatase-positive multinucleated bone-resorbing osteoclasts. Co-cultures of 5TGM1 with ST2 showed increased production of bone-resorbing activity and neutralizing antibodies against VCAM-1 or alpha(4)beta(1)-integrin inhibited this. The 5TGM1 cells contacting recombinant VCAM-1 produced increased osteoclastogenic and bone-resorbing activity. The activity was not blocked by the neutralizing antibody to known osteoclastogenic cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor, or parathyroid hormone-related peptide. These data suggest that myeloma cells are responsible for producing osteoclastogenic activity and that establishment of direct contact with marrow stromal cells via alpha(4)beta(1)-integrin/VCAM-1 increases the production of this activity by myeloma cells. They also suggest that the presence of stromal cells may provide a microenvironment that allows exclusive colonization of myeloma cells in the bone marrow. (Blood. 2000;96:1953-1960)

Absence of herpesvirus DNA sequences in the 5T murine model of human multiple myeloma.

Kaposi's sarcoma-associated herpesvirus (KSHV, also known as HHV-8) has been found in patients with multiple myeloma (MM) and postulated to be aetiologically associated with the development of this common plasma cell malignancy. A murine model of MM was previously established in which intravenous transfer of 5T myeloma cells into C57BL/KaLwRij mice resulted in characteristic features of human MM. In the present study, we sought to identify herpesvirus DNA sequences in this murine model of MM through polymerase chain reaction (PCR) analysis using primers specific for KSHV, murine herpesvirus 68 (MHV68) and murine cytomegalovirus (MCMV) as well as consensus primers designed from the highly conserved DNA polymerase genes of the Herpesviridae family. None of the DNA samples from whole bone marrow (n = 6) or dendritic cells enriched by long-term culture (n = 8) of 5T myeloma-bearing mice as well as the 5T myeloma cell lines (n = 3) maintained in long-term culture yielded specific amplification products in any of the PCR assays. Two KSHV-specific serological assays measuring antibodies to KSHV latent and lytic antigens also failed to detect the presence of anti-KSHV antibodies in mice that developed MM. These results suggest that the development of 5T murine MM is unlikely to be involved with KSHV or a KSHV-like murine herpesvirus.