Gonadal Hormones and Bone.

In both sexes, estrogen is one of the most essential hormones for maintaining bone integrity. Also, especially in men, androgen has beneficial effects on bone independent of estrogen. However, estrogen replacement therapy for postmenopausal women increases the risk of developing breast cancer and endometrial cancer, and androgen replacement therapy for partial androgen deficiency of the aging male increases the risk of developing prostate cancer. Various mechanisms have been proposed on the effects of gonadal hormones on bone, such as effects through cytokines including IL-6 and effects on the OPG/RANKL ratio. In addition, large amounts of new information deriving from high-throughput gene expression analysis raise the possibility of multiple other effects on bone cells. Both estrogen and androgen exert their effects via the estrogen receptor (ER) or the androgen receptor (AR), which belongs to the nuclear receptor superfamily. Compounds such as selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) also bind ER and AR, respectively. However, SERMs and SARMs alter the ER or AR structure differently from estrogen or androgen, resulting in other downstream gene responses. As a result they can exert favorable effects on bone while suppressing the undesirable actions of estrogen and androgen. Elucidation of ER and AR ligand-specific and tissue-specific gene regulation mechanisms will also provide information on the signal transduction mechanisms of other nuclear receptors and will be valuable for the development of new therapeutic agents.

Dentin phosphoprotein binds annexin 2 and is involved in calcium transport in rat kidney ureteric bud cells.

Dentin phosphoprotein (DPP) is the most abundant noncollagenous protein in the dentin, where it plays a major role in the mineralization of dentin. However, we and others have shown that in addition to being present in the dentin, DPP is also present in nonmineralizing tissues like the kidney, lung, and salivary glands, where it conceivably has other functions such as in calcium transport. Because annexins have been implicated as calcium transporters, we examined the relationships between DPP and annexins. In this report, we show that DPP binds to annexin 2 and 6 present in a rat ureteric bud cell line (RUB1). Immunofluorescence studies show that annexin 2 and DPP colocalize in these cells. In addition, DPP and annexin 2 colocalize in the ureteric bud branches of embryonic metanephric kidney. In the RUB1 cells and ureteric bud branches of embryonic kidney, colocalization was restricted to the cell membrane. Studies on calcium influx into RUB cells show that in the presence of anti-DPP, there was a 40% reduction of calcium influx into these cells. We postulate that DPP has different functions in the kidney as compared with the odontoblasts. In the odontoblasts, its primary function is in the extracellular mineralization of dentin, whereas in the kidney it may participate in calcium transport.

Antitumor agent cabozantinib decreases RANKL expression in osteoblastic cells and inhibits osteoclastogenesis and PTHrP-stimulated bone resorption.

Cabozantinib, an inhibitor of vascular endothelial growth factor and hepatocyte growth factor signaling, decreases bone lesions in patients with prostate cancer. To determine direct effects of cabozantinib on bone, resorption in neonatal mouse bone organ culture and on gene expression, proliferation, and phenotypic markers in osteoblast and osteoclast cell lines were examined. Cabozantinib, 0.3 and 3 µM, prevented PTHrP-stimulated calcium release from neonatal mouse calvaria. Since the effect on resorption could reflect effects on osteoblasts to prevent osteoclast activation, or direct inhibition of osteoclasts, responses in osteoblastic and osteoclast precursor cell lines were examined. Twenty-four-hour treatment of osteoblastic MC3T3-E1 cells with 3 µM cabozantinib decreased expression of receptor activator of NFkB ligand (RANKL) and alkaline phosphatase. Forty-eight-hour treatment of MC3T3-E1 cells with 3 µM cabozantinib inhibited cell proliferation and decreased MTT activity. Effects on alkaline phosphatase activity were biphasic, with small stimulatory effects at concentrations below 3 µM. When RAW 264.7 osteoclast precursor cells differentiated with 20 ng/ml RANKL were co-treated for 24 h with 3 µM cabozantinib, expression of RANK, TRAP, cathepsin K, alpha v or beta 3 integrin, or NFATc1 were unaffected. Five-day treatment of RANKL-treated RAW 264.7 cells with 3 µM cabozantinib decreased TRAP and MTT activity. The results suggest that the osteoblast could be the initial target, with subsequent direct and indirect effects on osteoclastogenesis leading to decreased resorption. The multiple effects of cabozantinib on the cell microenvironment of bone are consistent with its effectiveness in reducing lesions from prostate cancer metastases.

Regulation of breast cancer-induced bone lesions by ß-catenin protein signaling.

Breast cancer patients have an extremely high rate of bone metastases. Morphological analyses of the bones in most of the patients have revealed the mixed bone lesions, comprising both osteolytic and osteoblastic elements. ß-Catenin plays a key role in both embryonic skeletogenesis and postnatal bone regeneration. Although this pathway is also involved in many bone malignancy, such as osteosarcoma and prostate cancer-induced bone metastases, its regulation of breast cancer bone metastases remains unknown. Here, we provide evidence that the ß-catenin signaling pathway has a significant impact on the bone lesion phenotype. In this study, we established a novel mouse model of mixed bone lesions using intratibial injection of TM40D-MB cells, a breast cancer cell line that is highly metastatic to bone. We found that both upstream and downstream molecules of the ß-catenin pathway are up-regulated in TM40D-MB cells compared with non-bone metastatic TM40D cells. TM40D-MB cells also have a higher T cell factor (TCF) reporter activity than TM40D cells. Inactivation of ß-catenin in TM40D-MB cells through expression of a dominant negative TCF4 not only increases osteoclast differentiation in a tumor-bone co-culture system and enhances osteolytic bone destruction in mice, but also inhibits osteoblast differentiation. Surprisingly, although tumor cells overexpressing ß-catenin did induce a slight increase of osteoblast differentiation in vitro, these cells display a minimal effect on osteoblastic bone formation in mice. These data collectively demonstrate that ß-catenin acts as an important determinant in mixed bone lesions, especially in controlling osteoblastic effect within tumor-harboring bone environment.

Sex-specific effects of estrogen and androgen on gene expression in human monocyte-derived osteoclasts.

Estrogen and androgen are both critical for the maintenance of bone, but the target cells, mechanisms, and responses could be sex-specific. To compare sex-specific actions of estrogen and androgen on osteoclasts, human peripheral blood mononuclear precursor cells from adult Caucasian males (n = 3) and females (n = 3) were differentiated into osteoclasts and then treated for 24 h with 17ß-estradiol (10 nM) or testosterone (10 nM). Gene expression was studied with a custom designed qPCR-based array containing 94 target genes related to bone and hormone action. In untreated osteoclasts, 4 genes showed significant gender differences. 17ß-estradiol significantly affected 12 genes in osteoclasts from females and 6 genes in osteoclasts from males. Fifteen of the 18 17ß-estradiol-responsive genes were different in the cells from the two sexes; 2 genes affected by 17ß-estradiol in both sexes were regulated oppositely in the two sexes. Testosterone significantly affected 6 genes in osteoclasts from females and 2 genes in osteoclasts from males; all except one were different in the two sexes. 17ß-estradiol and testosterone largely affected different genes, suggesting that conversion of testosterone to 17ß-estradiol had a limited role in the responses. The findings indicate that although osteoclasts from both sexes respond to 17ß-estradiol and testosterone, the effects of both 17ß-estradiol and testosterone differ in the two sexes, highlighting the importance of considering gender in the design of therapy.

Osteoclastogenic activity and RANKL expression are inhibited in osteoblastic cells expressing constitutively active Gα(12) or constitutively active RhoA.

Gα(12)-RhoA signaling is a parathyroid hormone (PTH)-stimulated pathway that mediates effects in bone and may influence genetic susceptibility to osteoporosis. To further elucidate effects of the pathway in osteoblasts, UMR-106 osteoblastic cells were stably transfected with constitutively active (ca) Gα(12) or caRhoA or dominant negative (dn) RhoA and co-cultured with RAW 264.7 cells to determine effects on hormone-stimulated osteoclastogenesis. Whereas PTH and calcitriol-stimulated osteoclastogenesis in co-cultures with UMR-106 cells expressing pcDNA or dominant negative RhoA, the osteoclastogenic effects of PTH and calcitriol were significantly attenuated when the UMR-106 cells expressed either caRhoA or caGα(12). These inhibitory effects were partially reversed by the Rho kinase inhibitor Y27632. None of the constructs affected osteoclastogenesis in untreated co-cultures, and the constructs did not inhibit the osteoclastogenic responses to receptor activator of NFκB ligand (RANKL). To investigate the mechanism of the inhibitory effects of caGα(12) and caRhoA, expression of RANKL, osteoprotegerin (OPG), osteopontin (OPN), and intercellular adhesion molecule-1 (ICAM) in response to PTH or calcitriol was examined in the UMR-106 cells. In the cells expressing pcDNA or dnRhoA, PTH and calcitriol increased RANKL mRNA and decreased OPG mRNA, whereas these effects were absent in the cells expressing caGα(12) or caRhoA. Basal expression of RANKL and OPG was unaffected by the constructs. The results suggest that Gα(12)-RhoA signaling can inhibit hormone-stimulated osteoclastogenesis by effects on expression of RANKL and OPG. Since PTH can stimulate the Gα(12)-RhoA pathway, the current findings could represent a homeostatic mechanism for regulating osteoclastogenic action.

The small GTPase RhoA is crucial for MC3T3-E1 osteoblastic cell survival.

Prolongation of cell survival through prevention of apoptosis is considered to be a significant factor leading to anabolic responses in bone. The current studies were carried out to determine the role of the small GTPase, RhoA, in osteoblast apoptosis, since RhoA has been found to be critical for cell survival in other tissues. We investigated the effects of inhibitors and activators of RhoA signaling on osteoblast apoptosis. In addition, we assessed the relationship of this pathway to parathyroid hormone (PTH) effects on apoptotic signaling and cell survival. RhoA is activated by geranylgeranylation, which promotes its membrane anchoring. In serum-starved MC3T3-E1 osteoblastic cells, inhibition of geranylgeranylation with geranylgeranyl transferase I inhibitors increased activity of caspase-3, a component step in the apoptosis cascade, and increased cell death. Dominant negative RhoA and Y27632, an inhibitor of the RhoA effector Rho kinase, also increased caspase-3 activity. A geranylgeranyl group donor, geranylgeraniol, antagonized the effect of the geranylgeranyl transferase I inhibitor GGTI-2166, but could not overcome the effect of the Rho kinase inhibitor. PTH 1-34, a potent anti-apoptotic agent, completely antagonized the stimulatory effects of GGTI-2166, dominant negative RhoA, and Y27632, on caspase-3 activity. The results suggest that RhoA signaling is essential for osteoblastic cell survival but that the survival effects of PTH 1-34 are independent of this pathway.

Direct inhibitory and indirect stimulatory effects of RAGE ligand S100 on sRANKL-induced osteoclastogenesis.

Diabetes results in increased fracture risk, and advance glycation endproducts (AGEs) have been implicated in this pathophysiology. S100 proteins are ligands for the receptor of AGEs (RAGE). An intracellular role of the S100 family member S100A4 (Mts1) to suppress mineralization has been described in pre-osteoblastic MC3T3-E1 cells. However, S100 proteins could have additional effects on bone. The goal of the current study was to determine effects of increased extracellular S100 on osteoclastogenesis. We first determined the direct effects of S100 on pre-osteoclast proliferation and osteoclastic differentiation. RANKL-treated RAW 264.7 cell proliferation and TRAP activity were significantly inhibited by S100, and the number and size of TRAP-positive multinucleated cells were decreased. We then determined whether S100 could affect osteoclastogenesis by an indirect process by examining effects of conditioned media from S100-treated MC3T3-E1 cells on osteoclastogenesis. In contrast to the direct inhibitory effect of S100, the conditioned media promoted RAW 264.7 cell proliferation and TRAP activity, with a trend toward increased TRAP-positive multinucleated cells. S100 treatment of the MC3T3-E1 cells for 14 days did not significantly affect alkaline phosphatase, M-CSF, or OPG gene expression. RANKL was undetectable in both untreated and treated cells. The treatment slightly decreased MC3T3-E1 cell proliferation. Interestingly, S100 treatment increased expression of RAGE by the MC3T3-E1 cells. This suggested the possibility that S100 could increase soluble RAGE, which acts as a decoy receptor for S100. This decrease in availability of S100, an inhibitor of pre-osteoclast proliferation, could contribute to osteoclastogenesis, ultimately resulting in increased bone resorption.

Inhibitory effects of mevastatin and a geranylgeranyl transferase I inhibitor (GGTI-2166) on mononuclear osteoclast formation induced by receptor activator of NF kappa B ligand (RANKL) or tumor necrosis factor-alpha (TNF-alpha).

We have previously reported that the statin mevastatin (compactin) reversibly inhibits the fusion of TRAP-positive mononuclear preosteoclasts (pOCs) into multinucleated osteoclasts and disrupts the actin ring in mature osteoclasts through the inhibition of protein prenylation. Protein geranylgeranylation, specifically, is known to be required for pOC fusion and for the function and survival of mature osteoclasts. However, it has not been determined whether protein geranylgeranylation is involved in early differentiation of osteoclasts (pOC formation). The current study shows that statins and the geranylgeranyl transferase I inhibitor GGTI-2166 inhibit the pOC formation induced by RANKL or TNF-alpha in cultures of both mouse marrow-derived macrophage-colony-stimulating factor (M-CSF) dependent monocytes (MD cells) and the mouse monocyte cell line RAW 264.7 (RAW cells). Mevastatin, 0.1-0.6 microM, inhibited the formation of pOCs induced by receptor activator of nuclear factor-kappaB ligand (RANKL) or tumor necrosis factor (TNF-alpha) in both cell cultures. The inhibitory effects of mevastatin were overcome by the addition of mevalonate, farnesyl pyrophosphate or geranylgeranyl pyrophosphate. GGTI-2166 inhibited TRAP activity induced by RANKL or TNF-alpha in both cell cultures and prevented the incorporation of [3H]all-trans geranylgeraniol into prenylated proteins in RAW cells. However, the farnesyl transferase inhibitor FTI-2153 did not inhibit TRAP activity although FTI prevented the incorporation of [14C]mevalonate into farnesylated proteins in RAW cells. Clostridium difficile cytotoxin B (toxin B) inhibited pOC formation induced by RANKL or TNF-alpha in both cell cultures. The inhibitory effects of statins and GGTI-2166 on pOC formation may result from the inhibition of the geranylgeranylation of G-proteins, such as Rho or Rac, suggesting that the geranylgeranylation of these proteins is involved in the early differentiation of progenitor cells into pOCs.

Role of protein kinase A, phospholipase C and phospholipase D in parathyroid hormone receptor regulation of protein kinase Calpha and interleukin-6 in UMR-106 osteoblastic cells.

Parathyroid hormone (PTH) stimulates both bone formation and resorption by activating diverse osteoblast signalling pathways. Upstream signalling for PTH stimulation of protein kinase C-alpha (PKCalpha) membrane translocation and subsequent expression of the pro-resorptive cytokine interleukin-6 (IL-6) was investigated in UMR-106 osteoblastic cells. PTH 1-34, PTH 3-34, PTHrP and PTH 1-31 stimulated PKCalpha translocation and IL-6 promoter activity. Pharmacologic intervention at the adenylyl cyclase (AC) pathway (forskolin, IBMX, PKI) failed to alter PTH 1-34- or PTH 3-34-stimulated PKCalpha translocation. The phosphoinositol-phospholipase C (PI-PLC) antagonist U73122 slightly decreased PTH 1-34-stimulated PKCalpha translocation; however, the control analogue U73343 acted similarly. Propranolol, an inhibitor of phosphatidic acid (PA) phosphohydrolase, decreased diacylglycerol (DAG) formation and attenuated PTH 1-34- and PTH 3-34-stimulated PKCalpha translocation and IL-6 promoter activity, suggesting a phospholipase D (PLD)-dependent mechanism. This is the first demonstration that PLD-mediated signalling leads to both PKC-alpha translocation and IL-6 promoter activation in osteoblastic cells.

Parathyroid hormone stimulates phosphatidylethanolamine hydrolysis by phospholipase D in osteoblastic cells.

Parathyroid hormone (PTH) and phorbol-12,13-dibutyrate (PDBu) stimulate phospholipase D (PLD) activity and PC hydrolysis in UMR-106 osteoblastic cells {Singh, A.T., Kunnel, J.G., Strieleman, P.J., and Stern, P.H. (1999) Parathyroid Hormone (PTH)-(1-34), [Nle8,18,Tyr34]PTH-(3-34) Amide, PTH-(1-31) Amide, and PTH-Related Peptide-(1-34) Stimulate Phosphatidylcholine Hydrolysis in UMR-106 Osteoblastic Cells: Comparison with Effects of Phorbol 12,13-Dibutyrate, Endocrinology 140, 131-137}. The current studies were designed to determine whether ethanolamine-containing phospholipids, and specifically PE, could also be substrates. In cells labeled with 14C-ethanolamine, PTH and PDBu treatment decreased 14C-PE. In cells co-labeled with 3H-choline and 14C-ethanolamine, PTH and PDBu treatment increased both 3H-choline and 14C-ethanolamine release from the cells. Choline and ethanolamine phospholipid hydrolysis was increased within 5 min, and responses were sustained for at least 60 min. Maximal effects were obtained with 10 nM PTH and 50 nM PDBu. Dominant negative PLD1 and PLD2 constructs inhibited the effects of PTH on the phospholipid hydrolysis. The results suggest that both PC and PE are substrates for phospholipase D in UMR-106 osteoblastic cells and could therefore be sources of phospholipid hydrolysis products for downstream signaling in osteoblasts.

Rho and Rho kinase are involved in parathyroid hormone-stimulated protein kinase C alpha translocation and IL-6 promoter activity in osteoblastic cells.

UNLABELLED: The role of small G-proteins in PTH-stimulated PKC translocation and IL-6 promoter expression in UMR-106 cells was determined. The effects of PTH(1-34) and PTH(3-34) in stimulating PKCalpha translocation and IL-6 were inhibited by agents that interfere with the activity of small G-proteins of the Rho family and with the downstream kinase Rho kinase. INTRODUCTION: Activation of protein kinase C (PKC) is a signaling mechanism by which parathyroid hormone (PTH) modulates interleukin-6 (IL-6) in osteoblasts, leading to osteoclastogenesis and bone resorption. PKCalpha and PKCbetaI are translocated after treatment with PTH in UMR-106 osteoblastic cells; however, the pathway leading to PKC isozyme translocation is not established. Diacylglycerol (DAG) generation from phospholipase D (PLD) is one pathway of PKC activation, and PTH-mediated PLD activity is dependent on small G-proteins of the Rho family. This study investigated whether Rho proteins modulate the PKCalpha translocation and IL-6 promoter activity stimulated by PTH in UMR-106 cells. MATERIALS AND METHODS: UMR-106 cells were treated with PTH(1-34) or PTH(3-34). PKC translocation was determined by immunofluorescence, Rho A activation by Rhotekin assay and by translocation assessed by Western blotting in membrane and cytosol fractions, and IL-6 promoter expression by luciferase assay. RESULTS AND CONCLUSIONS: Inhibition of Rho proteins with Clostridium difficile toxin B or inhibition of Rho prenylation with GGTI attenuated PTH(1-34)- and PTH(3-34)-stimulated translocation of endogenous PKCalpha and IL-6 promoter activity. Expression of a constitutively active RhoA (RhoA63L) mimicked the effect of PTH(1-34) or PTH(3-34) to promote membrane localization of PKCalpha, whereas cells expressing a dominant negative RhoA (RhoA19N) did not respond to PTH(1-34) or PTH(3-34). The Rho kinase inhibitor Y27632 attenuated PTH(1-34)- and PTH(3-34)-stimulated PKCalpha translocation and IL-6 promoter activation. Rho seemed to be acting at a step before production of diacylglycerol (DAG), because the stimulation of PKCalpha translocation by the DAG mimetic phorbol 12,13 dibutyrate (PDBu) was unaffected by C. difficile toxin B or Y27632. These results indicate that Rho proteins are an important component of PTH signaling in osteoblastic cells and provide further demonstration of convergence between PKC and small G-protein signaling pathways.

Regulation of parathyroid hormone-stimulated phospholipase D in UMR-106 cells by calcium, MAP kinase, and small G proteins.

UNLABELLED: Signaling intermediates for PTH and phorbol activation of PLD in UMR-106 cells were determined. Calcium was required, and the effects of PTH, phorbol, and calcium were dependent on p42/44 MAP kinase and small G proteins, specifically RhoA, acting through Rho kinase. INTRODUCTION: Phospholipase D (PLD) plays a key signaling role in numerous cellular processes. PLD-stimulated hydrolysis of phosphatidylcholine (PC) generates phosphatidic acid, a source of diacylglycerol (DAG). We previously reported that parathyroid hormone (PTH) stimulates PLD activity in UMR-106 osteoblastic cells by a protein kinase C (PKC)-independent mechanism. The current study investigated the roles of calcium, MAP kinase, and small G proteins in PTH- and phorbol-12,13-dibutyrate (PDBu)-stimulated transphosphatidylation of ethanol, a reaction catalyzed by PLD. METHODS: UMR-106 cells were labeled with 3H-palmitic and treated in the presence of ethanol. Phosphatidylethanol was separated by thin-layer chromatography and detected by autoradiography, and the bands were scraped and counted. Statistical significance of the responses from three to nine replicates was determined by ANOVA and Tukey's post-test. RESULTS AND CONCLUSIONS: PTH and PDBu effects were attenuated by EGTA, BAPTA, nifedipine, and dantrolene, whereas ionomycin or 2X calcium increased basal PLD activity. PTH activated p42/p44 MAP kinase, and the effects of PTH, PDBu, and ionomycin on PLD, but not on calcium influx, were prevented by the MEK inhibitors PD98059 and U0126. Small G proteins were shown to be involved in the effects of PTH, PDBu, and ionomycin on PLD. Inhibition of ARF by brefeldin prevented the PLD activation by all three agonists. A nonselective Rho/Rac/cdc-42 inhibitor, Clostridium difficile toxin B, also inhibited the effects of all three agonists on PLD. More selective inhibition of RhoA with a dominant negative RhoA construct or by inhibiting geranylgeranyltransferase I antagonized the effects of PTH, PDBu, and ionomycin, as did inhibiting the downstream kinase, Rho kinase. The current results reveal the importance of calcium, MAP kinase, and small G proteins in PTH and PDBu stimulation of PLD activity in UMR-106 cells.

Effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on bone resorption and osteoclastogenesis in vitro.

The effects of an important new anti-inflammatory agent, the selective cyclooxygenase-2 inhibitor celecoxib, on bone resorption and osteoclastogenesis elicited by the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), the endotoxin lipopolysaccharide (LPS), and the systemic hormones 1alpha,25-dihydroxyvitamin D(3) and parathyroid hormone were examined in vitro. Bone resorption was evaluated by measuring calcium released into the culture medium in a neonatal mouse calvarial bone organ culture. Osteoclastogenesis was evaluated by measuring tartrate-resistant acid phosphatase activity in the cells in cocultures of bone marrow cells and osteoblastic cells and in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. Celecoxib (0.1 microM) completely inhibited the calcium release induced by IL-1beta, TNF-alpha, and LPS. The resorptive effect of 1alpha,25-dihydroxyvitamin D(3) was inhibited partially by celecoxib. In contrast, celecoxib did not inhibit the calcium release elicited by parathyroid hormone or prostaglandin E(2). Celecoxib (0.1 microM) also markedly inhibited osteoclastogenesis induced by these stimulators of bone resorption except for PGE(2) in the coculture system, whereas it failed to inhibit osteoclastogenesis in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. These results indicate that, under certain conditions, cyclooxygenase-2-dependent prostaglandin synthesis is critical for the bone resorption induced by IL-1beta, TNF-alpha, and LPS, and for the osteoclastogenesis induced by these pro-inflammatory molecules and calciotropic hormones. The prevention of prostaglandin synthesis by inflammatory cytokines in bone cells could contribute to the efficacy of celecoxib in preventing bone loss in rheumatoid arthritis.

Bisphosphonates and nonhealing femoral fractures: analysis of the FDA Adverse Event Reporting System (FAERS) and international safety efforts: a systematic review from the Research on Adverse Drug Events And Reports (RADAR) project.

BACKGROUND: In the United States, hip fracture rates have declined by 30% coincident with bisphosphonate use. However, bisphosphonates are associated with sporadic cases of atypical femoral fracture. Atypical femoral fractures are usually atraumatic, may be bilateral, are occasionally preceded by prodromal thigh pain, and may have delayed fracture-healing. This study assessed the occurrence of bisphosphonate-associated nonhealing femoral fractures through a review of data from the U.S. FDA (Food and Drug Administration) Adverse Event Reporting System (FAERS) (1996 to 2011), published case reports, and international safety efforts. METHODS: We analyzed the FAERS database with use of the proportional reporting ratio (PRR) and empiric Bayesian geometric mean (EBGM) techniques to assess whether a safety signal existed. Additionally, we conducted a systematic literature review (1990 to February 2012). RESULTS: The analysis of the FAERS database indicated a PRR of 4.51 (95% confidence interval [CI], 3.44 to 5.92) for bisphosphonate use and nonhealing femoral fractures. Most cases (n = 317) were attributed to use of alendronate (PRR = 3.32; 95% CI, 2.71 to 4.17). In 2008, international safety agencies issued warnings and required label changes. In 2010, the FDA issued a safety notification, and the American Society for Bone and Mineral Research (ASBMR) issued recommendations about bisphosphonate-associated atypical femoral fractures. CONCLUSIONS: Nonhealing femoral fractures are unusual adverse drug reactions associated with bisphosphonate use, as up to 26% of published cases of atypical femoral fractures exhibited delayed healing or nonhealing.

How vitamin D works on bone.

Vitamin D is important for the normal development and maintenance of bone. The elucidation of the vitamin D activation pathway and the cloning of the vitamin D receptor have advanced our understanding of the actions of vitamin D on bone. The preponderance of evidence indicates that 1,25(OH)2D3 enhances bone mineralization through its effects to promote calcium and phosphate absorption. Although 1,25(OH)2D3 stimulates bone resorption in vitro, treatment in vivo can prevent bone loss and fracture through several potential mechanisms. The development of vitamin D analogues has provided new therapeutic options for increasing bone mineral density and reducing fractures.

Systemic delivery of oncolytic adenoviruses targeting transforming growth factor-ß inhibits established bone metastasis in a prostate cancer mouse model.

We have examined whether Ad.sTßRFc and TAd.sTßRFc, two oncolytic viruses expressing soluble transforming growth factor-ß receptor II fused with human Fc (sTGFßRIIFc), can be developed to treat bone metastasis of prostate cancer. Incubation of PC-3 and DU-145 prostate tumor cells with Ad.sTßRFc and TAd.sTßRFc produced sTGFßRIIFc and viral replication; sTGFßRIIFc caused inhibition of TGF-ß-mediated SMAD2 and SMAD3 phosphorylation. Ad(E1-).sTßRFc, an E1(-) adenovirus, produced sTGFßRIIFc but failed to replicate in tumor cells. To examine the antitumor response of adenoviral vectors, PC-3-luc cells were injected into the left heart ventricle of nude mice. On day 9, mice were subjected to whole-body bioluminescence imaging (BLI). Mice bearing hind-limb tumors were administered viral vectors via the tail vein on days 10, 13, and 17 (2.5×10(10) viral particles per injection per mouse, each injection in a 0.1-ml volume), and subjected to BLI and X-ray radiography weekly until day 53. Ad.sTßRFc, TAd.sTßRFc, and Ad(E1-).sTßRFc caused significant inhibition of tumor growth; however, Ad.sTßRFc was the most effective among all the vectors. Only Ad.sTßRFc and TAd.sTßRFc inhibited tumor-induced hypercalcemia. Histomorphometric and synchrotron micro-computed tomographic analysis of isolated bones indicated that Ad.sTßRFc induced significant reduction in tumor burden, osteoclast number, and trabecular and cortical bone destruction. These studies suggest that Ad.sTßRFc and TAd.sTßRFc can be developed as potential new therapies for prostate cancer bone metastasis.