Enhanced Bone Resorption Research Articles

Inhibition of RANK/RANKL signal transduction pathway: A promising approach for osteoporosis treatment

Osteoporosis is a bone disease causing impaired bone strength. It is characterized by increased osteoclast formation or enhanced bone resorption, leading to an increased risk of fragility fractures. Its prevalence increases with age. The advent of an aging population suggests that progressively more individuals will develop this disease in the aging population. A number of drugs for the prevention and treatment of osteoporosis act by inhibiting bone resorption. However, the effectiveness of osteoporosis treatment in clinical practice is limited. Since the osteoclast is the only cell in the body that is capable of resorbing bone, understanding its biology will be necessary for developing a new therapeutic approach for osteoporosis. Recently, it was discovered that the receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system is an important signal transduction pathway that regulates osteoclast formation. The binding of OPG to RANKL inhibits the binding between RANKL and RANK; this, in turn, prevents osteoclast precursors from differentiating and fusing to form mature osteoclasts. Therefore, the inhibition of the RANK/RANKL pathway inhibits osteoclast formation, differentiation, activation, and bone resorption. A potential clinical antiresorptive therapy can be developed by using an anti-RANKL monoclonal antibody, such as denosumab, that binds to RANKL with high affinity and specificity and blocks RANKL-RANK interactions.

Mechanisms for formation of myeloma bone disease

Myeloma cells stimulate bone resorption by enhancing osteoclast formation and suppress bone formation by inhibiting osteoblast differentiation. Macrophage inflammatory protein (MIP)-1alpha and MIP-1beta as well as RANK ligand play a major role in the enhancement of bone resorption in myeloma. Myeloma cell-derived soluble Wnt inhibitors as well as TGF-beta released from the bone tissues through enhanced bone resorption are thought to suppress osteoblast differentiation. Such pathognomonically skewed cellular components in the bone marrow create a microenvironment suitable for myeloma cell growth and survival (a myeloma niche) , which should be targeted to suppress myeloma expansion along with amelioration of bone lesions.

Kinin B1 and B2 receptor expression in osteoblasts and fibroblasts is enhanced by interleukin-1 and tumour necrosis factor-α. Effects dependent on activation of NF-κB and MAP kinases

Pro-inflammatory mediators formed by the kallikrein–kinin system can stimulate bone resorption and synergistically potentiate bone resorption induced by IL-1 and TNF-α. We have shown that the effect is associated with synergistically enhanced RANKL expression and enhanced prostaglandin biosynthesis, due to increased cyclooxygenase-2 expression. In the present study, the effects of osteotropic cytokines and different kinins on the expression of receptor subtypes for bradykinin (BK), des-Arg10-Lys-BK (DALBK), IL-1β and TNF-α have been investigated. IL-1β and TNF-α enhanced kinin B1 and B2 receptor binding in the human osteoblastic cell line MG-63 and the mRNA expression of B1 and B2 receptors in MG-63 cells, human gingival fibroblasts and intact mouse calvarial bones. Kinins did not affect mRNA expression of IL-1 or TNF receptors. EMSA showed that IL-1β and TNF-α activated NF-κB and AP-1 in MG-63 cells. IL-1β stimulated NF-κB via a non-canonical pathway (p52/p65) and TNF-α via the canonical pathway (p50/p65). Activation of AP-1 involved c-Jun in both IL-1β and TNF-α stimulated cells, but c-Fos only in TNF-α stimulated cells. Phospho-ELISA and Western blots showed that IL-1β activated JNK and p38, but not ERK 1/2 MAP kinase. Pharmacological inhibitors showed that NF-κB, p38 and JNK were important for IL-1β induced stimulation of B1 receptors, and NF-κB and p38 for B2 receptors. p38 and JNK were important for TNF-α induced stimulation of B1 receptors, whereas NF-κB, p38 and JNK were involved in TNF-α induced expression of B2 receptors. These data show that IL-1β and TNF-α upregulate B1 and B2 receptor expression by mechanisms involving activation of both NF-κB and MAP kinase pathways, but that signal transduction pathways are different for IL-1β and TNF-α. The enhanced kinin receptor expression induced by the pro-inflammatory cytokines IL-1β and TNF-α might be one important mechanism involved in the synergistic enhancement of prostaglandin formation caused by co-treatment with kinins and one of the two cytokines. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including periodontitis and rheumatoid arthritis.

Aging and ovariectomy alter endothelium‐dependent and –independent vasodilation in the femoral principal nutrient artery

Enhanced bone resorption and bone blood flow (BBF) occurs subsequent to ovariectomy (OVX) whereas BBF declines with age. The potential of the osseous resistance vasculature to alter BBF in postmenopausal and age‐related osteoporosis is unclear. PURPOSE: We studied the effects of age, gender, and estrogen status on endothelium‐dependent and ‐independent vasodilation of the femoral principal nutrient artery (PNA). METHODS: INTACT female (6‐, 14‐, and 24‐ mon), OVX female (6‐, 14‐, and 24‐ mon), and male (6‐ and 24‐ mon) Fischer‐344 rats were studied. PNAs were isolated and cannulated. Endothelium‐dependent (ACh: 10−9 – 10−4 M) and ‐independent (DEA NONOate: 10−10 – 10−4 M) vasodilation were assessed. RESULTS: Declines in endothelial function occurred in aged rats but no differences in gender were observed. Endothelium‐dependent vasodilation in young or middle‐aged rats did not change following OVX. In aged OVX rats, vasodilation was enhanced to similar values observed in young INTACT and young OVX rats. Endothelium‐independent vasodilation was not altered with age or gender except in middle aged OVX rats vs. their INTACT controls. DISCUSSION: These data demonstrate that OVX influences both endothelium‐dependent and –independent vasodilator responses of the osseous resistance vasculature. Supported by the NIH Grant #1 R01 HL077224‐01 (JMD) and the NHLBI Postdoctoral Supplement (RDP).

JunD suppresses bone formation and contributes to low bone mass induced by estrogen depletion

JunD is an activator protein-1 (AP-1) component though its function in skeletal system is still not fully understood. To elucidate the role of JunD in the regulation of bone metabolism, we analyzed JunD-deficient mice. JunD deficiency significantly increased bone mass and trabecular number. This bone mass enhancement was due to JunD deficiency-induced increase in bone formation activities in vivo. Such augmentation of bone formation was associated with simultaneous increase in bone resorption while the former was dominant over the latter as accumulation of bone mass occurred in JunD-deficient mice. In a pathological condition relevant to postmenopausal osteoporosis, ovariectomy reduced bone mass in wild type (WT) mice as known before. Interestingly, JunD deficiency suppressed ovariectomy-induced increase in bone resorption and kept high bone mass. In addition, JunD deficiency also enhanced new bone formation after bone marrow ablation. Examination of molecular bases for these observations revealed that JunD deficiency enhanced expression levels of c-jun, fra-1, and fra-2 in bone in conjunction with elevated expression levels of runx2, type I collagen, and osteocalcin. Thus, JunD is involved in estrogen depletion-induced osteopenia via its action to suppress bone formation and to enhance bone resorption.

Prolactin directly enhances bone turnover by raising osteoblast-expressed receptor activator of nuclear factor κB ligand/osteoprotegerin ratio

Hyperprolactinemia leads to high bone turnover as a result of enhanced bone formation and resorption. Although its osteopenic effect has long been explained as hyperprolactinemia-induced hypogonadism, identified prolactin (PRL) receptors in osteoblasts suggested a possible direct action of PRL on bone. In the present study, we found that hyperprolactinemia induced by anterior pituitary transplantation (AP), with or without ovariectomy (Ovx), had no detectable effect on bone mineral density and content measured by dual-energy X-ray absorptiometry (DXA). However, histomorphometric studies revealed increases in the osteoblast and osteoclast surfaces in the AP rats, but a decrease in the osteoblast surface in the AP + Ovx rats. The resorptive activity was predominant since bone volume and trabecular number were decreased, and the trabecular separation was increased in both groups. Estrogen supplement (E2) fully reversed the effect of estrogen depletion in the Ovx but not in the AP + Ovx rats. In contrast to the typical Ovx rats, bone formation and resorption became uncoupled in the AP + Ovx rats. Therefore, hyperprolactinemia was likely to have some estrogen-independent and/or direct actions on bone turnover. Osteoblast-expressed PRL receptor transcripts and proteins shown in the present study confirmed our hypothesis. Furthermore, we demonstrated that the osteoblast-like cells, MG-63, directly exposed to PRL exhibited lower expression of alkaline phosphatase and osteocalcin mRNA, and a decrease in alkaline phosphatase activity. The ratios of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) proteins were increased, indicating an increase in the osteoclastic bone resorption. The present data thus demonstrated that hyperprolactinemia could act directly on bone to stimulate bone turnover, with more influence on bone resorption than formation. PRL enhanced bone resorption in part by increasing RANKL and decreasing OPG expressions by osteoblasts.

Multiple Myeloma Cell-Osteoblast Interaction Results in Impaired Bone Formation.

Osteolytic bone disease is a common complication of multiple myeloma (MM) resulting from uncoupled bone remodeling due to enhanced bone resorption and reduced bone formation. Bone formation is a complex process requiring functionally mature osteoblasts (OB). Mesenchymal stem cells differentiate into mature OB and following an active period of bone matrix synthesis lasting 1–2 weeks, they finally differentiate into inactive bone-lining cells or osteocytes. Although several studies have demonstrated that MM cells inhibit osteoblastogenesis via secretion of DKK1, a Wnt-pathway antagonist, the functional sequelae of interaction of mature OB with MM cells remains to be elucidated. Here, we studied the morphological and functional consequences induced by MM cells interacting with mature OB. Mature OB were generated from MM patients' bone marrow mononuclear cells by cultivation in differentiation media consisting of αMEM with 20% fetal bovine serum, β-glycerol phosphate (2.16 mg/ml), ascorbic acid (0.05 mg/ml) and dexamethasone (10 nM). These mature OBs were alkaline phosphatase (ALP) positive and secreted and mineralized bone matrix, as demonstrated by Alizarin Red staining. MM cell lines INA6 and MM1.S were co-cultured with mature OB at a 5:1 ratio for 2, 4 and 7 days in OB differentiation media and bone marrow stromal cells (BMSC) were used as negative controls. After 4 days of co-culture, we observed phenotypic changes featured by acquisition of a spindle-like shape with reduced ALP staining in OB. In contrast, OB alone were intensely ALP-positive and cuboidal-shaped cells. Co-culture with INA-6 MM cells induced a reduction in ALP enzymatic activity in a time-dependent manner by 28% (± 10%) at day 2 and 72% (± 5%) at day 4 (p<0.05), respectively, whereas co-culture with MM1.S induced a 38% (± 5%) reduction after 4 days. Other MM cell lines induced similar effects. We then verified OB activity by assessing osteocalcin release and matrix mineralization. Importantly, osteocalcin secretion was completely abrogated in the presence of INA6, while MM1.S reduced it by 50% as early as day 2 (p<0.05). Moreover, Alizarin red staining demonstrated an impairment of matrix mineralization after 7 days of co-culture. Reduced OB function in the presence of MM cells was further confirmed by downregulation of Type-I collagen expression in OB. These effects were associated with only modest (10%) OB apoptosis as demonstrated by APO2.7 staining after 4 days of co-culture compared to OB alone. These phenotypic and functional sequelae on OB were not induced by co-culture supernatants, suggesting the requirement for direct MM cell/OB contact. These results therefore suggest that MM cell/mature OB interactions result in inhibition of bone formation by inactivation of mature OB. Ongoing studies are characterizing the mechanism by which MM cells induce OB inactivation and whether these changes affect the OC compartment. These studies of MM cell-OB interactions will form the basis for evaluation of novel agents with anabolic effects on the bone in the future.

The effects of TBC3214, a selective endothelin ETA receptor antagonist, on orthodontic tooth movement in rats

Many chemical messengers are involved in the process of alveolar bone and periodontal ligament remodelling during orthodontic tooth movement. Among them is probably endothelin-1 (ET-1). Its role in this process has been partly explained using tezosentan, which affects endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Tezosentan enhances orthodontic tooth movement. The aim of this study was to determine the possible effects of a highly selective ET(A) antagonist on orthodontic tooth movement in rats. Thirty male Wistar rats, 11-12 weeks of age, divided into three equal groups. In group I, a closed-coil spring was used and they were treated daily with 15 mg/kg body weight of TBC3214, a highly selective ET(A) antagonist. A closed-coil spring was also used in group II and the animals were treated daily with a placebo. Group III were treated daily with a placebo. The coil spring delivered a force of 25 cN and was attached between the upper left first molar and upper left incisor. The distance between the teeth was measured with a digital calliper (accuracy +/- 0.01 mm) on days 0, 7, 14, 21, 24, 32, 37, and 40. The differences in the distance between the teeth were calculated to determine the amount of tooth movement. Statistical analysis was performed using two-way analysis of variance, Bonferroni's correction, and paired t-tests. The distance between the upper left first molar and the upper left incisor decreased in groups I and II. In group I, tooth movement was significantly less on days 32 and 37 (P < 0.01) and on day 40 (P < 0.001) compared with group II. In group III, the distance between the teeth increased during the study (P < 0.001). In animals treated daily with TBC3214, tooth movement was significantly less compared with the animals treated with a placebo. It is concluded that ET-1, which is the predominant form of endothelin isopeptides, is involved in orthodontic tooth movement in rats, probably by enhancing bone resorption via ET(A) receptors.

Vascular calcification and arterial stiffness in chronic kidney disease: Implications and management

Cardiovascular (CV) disease is the commonest cause of mortality in patients with chronic kidney disease (CKD). Vascular calcification (VC), induced by calcium and phosphate excess and uraemia, is a major risk factor and is independently associated with CV events and death. Local and systemic calcium-regulatory proteins as well as inhibitory extracellular factors are involved in the pathogenesis of VC. In CKD the balance becomes dysregulated leading to differentiation of vascular smooth muscle cells into phenotypically distinct osteoblast-like cells with subsequent ossification of the arterial wall. Associated with imbalances in mineral metabolism, VC has intimate interactions with bone mineralization and enhanced bone resorption. Arterial stiffness represents the functional disturbance of VC, with reduced compliance of large arteries, and predominantly results from greater medial calcification. As with VC, arterial stiffness is an independent predictor of CV mortality and patients with CKD have greater arterial stiffness than the general population resulting in the principal consequences of left ventricular hypertrophy and altered coronary perfusion. Both VC and arterial stiffness can be measured through non-invasive techniques involving computed tomography, ultrasound, echocardiography, and pulse wave velocity. Management in CKD is difficult but detection, prevention and treatment is crucial to reduce CV mortality. The optimal control of mineral metabolism, especially hyperphosphatemia with non-calcium based phosphate binders, has been shown to be effective to reduce VC, and attenuation of arterial stiffness, especially with good blood pressure control, can have a favourable effect with regression of left ventricular hypertrophy. The use of bisphosphonates, calcimimetics, vitamin D therapy and newer experimental treatments, as well as nocturnal dialysis, may have potential benefit.

Bortezomib inhibits human osteoclastogenesis

In multiple myeloma, the overexpression of receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL) leads to the induction of NF-kappaB and activator protein-1 (AP-1)-related osteoclast activation and enhanced bone resorption. The purpose of this study was to examine the molecular and functional effects of proteasome inhibition in RANKL-induced osteoclastogenesis. Furthermore, we aimed to compare the outcome of proteasome versus selective NF-kappaB inhibition using bortezomib (PS-341) and I-kappaB kinase inhibitor PS-1145. Primary human osteoclasts were derived from CD14+ precursors in presence of RANKL and macrophage colony-stimulating factor (M-CSF). Both bortezomib and PS-1145 inhibited osteoclast differentiation in a dose- and time-dependent manner and furthermore, the bone resorption activity of osteoclasts. The mechanisms of action involved in early osteoclast differentiation were found to be related to the inhibition of p38 mitogen-activated protein kinase pathways, whereas the later phase of differentiation and activation occurred due to inhibition of p38, AP-1 and NF-kappaB activation. The AP-1 blockade contributed to significant reduction of osteoclastic vascular endothelial growth factor production. In conclusion, our data demonstrate that proteasomal inhibition should be considered as a novel therapeutic option of cancer-induced lytic bone disease.

Administration of Cyclooxygenase-2 Inhibitor Reduces Joint Inflammation but Exacerbates Osteopenia in IL-1α Transgenic Mice Due to GM-CSF Overproduction

IL-1alpha transgenic (Tg) mice exhibit chronic inflammatory arthritis and subsequent osteopenia, with IL-1-induced GM-CSF playing an important role in the pathogenesis. This study analyzed the mechanisms underlying osteopenia in Tg mice, and the therapeutic effects of the cyclooxygenase-2 inhibitor celecoxib on such osteopenia. Inhibited osteoclast formation was observed in RANKL-treated bone marrow cell (BMC) cultures from Tg mice and coculture of Tg-derived BMCs and wild-type-derived primary osteoblasts (POBs). FACS analysis indicated that this inhibition was attributable to a decreased number of osteoclast precursors within Tg-derived BMCs. Moreover, in coculture of Tg-derived POBs and either Tg- or wild-type-derived BMCs, osteoclast formation was markedly inhibited because Tg-derived POBs produced abundant GM-CSF, known as a potent inhibitor of osteoclast differentiation. Histomorphometric analysis of Tg mice revealed that both bone formation and resorption were decreased, with bone formation decreased more prominently. Interestingly, administration of celecoxib resulted in further deterioration of osteopenia where bone formation was markedly suppressed, whereas bone resorption remained unchanged. These results were explained by our in vitro observation that celecoxib dose-dependently and dramatically decreased osteogenesis by Tg mouse-derived POBs in culture, whereas mRNA expressions of GM-CSF and M-CSF remained unchanged. Consequently, blockade of PGE(2) may exert positive effects on excessively enhanced bone resorption observed in inflammatory bone disease, whereas negative effects may occur mainly through reduced bone formation, when bone resorption is constitutively down-regulated as seen in Tg mice.

Constitutive Activity of the Osteoblast Ca2+-Sensing Receptor Promotes Loss of Cancellous Bone

Changes in extracellular [Ca2+] modulate the function of bone cells in vitro via the extracellular Ca2+-sensing receptor (CaR). Within bone microenvironments, resorption increases extracellular [Ca2+] locally. To determine whether enhanced CaR signaling could modulate remodeling and thereby bone mass in vivo, we generated transgenic mice with a constitutively active mutant CaR (Act-CaR) targeted to their mature osteoblasts by the 3.5 kb osteocalcin promoter. Longitudinal microcomputed tomography of cancellous bone revealed reduced bone volume and density, accompanied by a diminished trabecular network, in the Act-CaR mice. The bone loss was secondary to an increased number and activity of osteoclasts, demonstrated by histomorphometry of secondary spongiosa. Histomorphometry, conversely, indicates that bone formation rates were unchanged in the transgenic mice. Constitutive signaling of the CaR in mature osteoblasts resulted in increased expression of RANK-L (receptor activator of nuclear factor-kappaB ligand), the major stimulator of osteoclast differentiation and activation, which is the likely underlying mechanism for the bone loss. The phenotype of Act-CaR mice is not attributable to systemic changes in serum [Ca2+] or PTH levels. We provide the first in vivo evidence that increased signaling by the CaR in mature osteoblasts can enhance bone resorption and further propose that fluctuations in the [Ca2+] within the bone microenvironment may modulate remodeling via the CaR.

Bradykinin potentiates cytokine‐induced prostaglandin biosynthesis in osteoblasts by enhanced expression of cyclooxygenase 2, resulting in increased RANKL expression

Bradykinin (BK) stimulates bone resorption in vitro and synergistically potentiates interleukin-1 (IL-1)-induced bone resorption and prostaglandin (PG) formation, suggesting that kinins are important in inflammation-induced bone loss. The present study was undertaken to study 1) the role of the kinin B1 and B2 receptors in the synergistic interaction with IL-1 and tumor necrosis factor alpha (TNFalpha), 2) the molecular mechanisms involved in synergistic enhancement of PG formation, and 3) the effects of kinins on cytokine-induced expression of RANKL, RANK, and osteoprotegerin (OPG) (the latter being crucial molecules in osteoclast differentiation). Formation of PGs, expression of enzymes involved in arachidonic acid metabolism, and expression of RANKL, RANK, and OPG were assessed in the human osteoblastic cell line MG-63 and in mouse calvarial bones. The role of NF-kappaB and MAP kinases was studied using pharmacologic inhibitors. PGE(2) formation and cyclooxygenase 2 (COX-2) protein expression were induced by IL-1beta and potentiated by kinins with affinity for the B1 or B2 receptors, resulting in PGE(2)-dependent enhancement of RANKL. The enhancements of PGE(2) formation and COX-2 were markedly decreased by inhibition of p38 and JNK MAP kinases, whereas inhibition of NF-kappaB resulted in abolishment of the PGE(2) response with only slight inhibition of COX-2. Kinin B1 and B2 receptors synergistically potentiate IL-1- and TNFalpha-induced PG biosynthesis in osteoblasts by a mechanism involving increased levels of COX-2, resulting in increased RANKL. The synergistic stimulation is dependent on NF-kappaB and MAP kinases. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including that in rheumatoid arthritis.

Gab2 plays distinct roles in bone homeostasis at different time points

Grb2-associated binder 2 (Gab2) is an adaptor molecule that can be tyrosine phosphorylated by various growth factors and cytokines. Gab2 is known to play a role in signaling pathways downstream of cytokines that regulate bone homeostasis, including M-CSF, RANKL, and IL-6. To clarify the role of Gab2 in bone homeostasis during distinct phases of skeletal development, we compared phenotypic changes in bone homeostasis in Gab2(-/-) mice at two different ages. Although Gab2(-/-) mice showed increased bone volume at both time points, the reasons underlying the increased bone volume differed. At 6 weeks, the increased bone volume was due to enhanced bone resorption and bone formation, indicating that Gab2 plays a negative regulatory role for both osteoclastogenesis and osteoblast differentiation. At 12 weeks, the increased bone volume resulted from reduced osteoclast differentiation, indicating that Gab2 plays a positive regulatory role for osteoclastogenesis. Thus, Gab2 plays opposite roles in osteoclastogenesis during the phases of skeletal development and maintenance.

Basic and clinical aspects of osteoporosis in inflammatory bowel disease

Low bone mineral density and the increased risk of fracture in gastrointestinal diseases have a multifactorial pathogenesis. Inflammatory bowel disease (IBD) has been associated with an increased risk of osteoporosis and osteopenia and epidemiologic studies have reported an increased prevalence of low bone mass in patients with IBD. Certainly, genetics play an important role, along with other factors such as systemic inflammation, malnutrition, hypogonadism, glucocorticoid therapy in IBD and other lifestyle factors. At a molecular level the proinflammatory cytokines that contribute to the intestinal immune response in IBD are known to enhance bone resorption. There are genes influencing osteoblast function and it is likely that LRP5 may be involved in the skeletal development. Also the identification of vitamin D receptors (VDRs) and some of its polymorphisms have led to consider the possible relationships between them and some autoimmune diseases and may be involved in the pathogenesis through the exertion of its immunomodulatory effects during inflammation. Trying to explain the physiopathology we have found that there is increasing evidence for the integration between systemic inflammation and bone loss likely mediated via receptor for activated nuclear factor kappa-B (RANK), RANK-ligand, and osteoprotegerin, proteins that can affect both osteoclastogenesis and T-cell activation. Although glucocorticoids can reduce mucosal and systemic inflammation, they have intrinsic qualities that negatively impact on bone mass. It is still controversial if all IBD patients should be screened, especially in patients with preexisting risk factors for bone disease. Available methods to measure BMD include single energy x-ray absorptiometry, DXA, quantitative computed tomography (QCT), radiographic absorptiometry, and ultrasound. DXA is the establish method to determine BMD, and routinely is measured in the hip and the lumbar spine. There are several treatments options that have proven their effectiveness, while new emergent therapies such as calcitonin and teriparatide among others remain to be assessed.

Biochemical approach to the detection and monitoring of metastatic bone disease: What do we know and what questions need answers?

Metastatic spread to bones frequently occurs in several types of cancer diseases, in particular breast, prostate, and lung cancer. Infiltration of bone by tumour cells is a source of several complications including severe bone pain, spinal cord compression, hypercalcemia, pathologic fractures, all reducing quality of life and worsening prognosis. Therefore, early recognition of bone metastases is among the highest priorities in the clinical management of cancer disease. Currently, detection and staging relies on radiological imaging techniques (scintigraphy, radiography, computer tomography, etc.). Due to their limited sensitivity and/or inconveniences, irradiation, and considerable costs related to serial use, they are not suited for close monitoring of cancer patients to capture skeletal spread in an early stage or to follow-up on therapeutical responses. Interaction of tumour cells with surrounding bone cells leads to enhanced bone resorption and/or bone formation. These cellular processes result in the release of numerous epitopes that, if detected by immunoassays, can reflect the changes of the rate of bone turnover and the occurrence of metastatic spread to bone. Numerous studies reported elevated levels of bone turnover markers in patients with bone metastases proportionally to the extent of skeletal involvement. Furthermore, preliminary data suggest that biomarkers can predict skeletal-related events (SREs), disease progression, and even cancer-related death. The present review intends to summarize the list of emerged biomarkers, major studies assessing their relative utility for detection of bone metastases in different types of cancer disease, and discuss their potentials for becoming part of screening protocols for improving our success rate in the early detection of metastatic bone disease.

Differential expression of receptor activator of nuclear factor‐κB ligand and osteoprotegerin mRNA in periodontal diseases

Receptor activator of nuclear factor-kappaB ligand (RANKL) is responsible for the induction of osteoclastogenesis and bone resorption, whereas its decoy receptor, osteoprotegerin, can directly block this action. Because this dyad of cytokines is crucial for regulating the bone remodelling process, imbalances in their expression may cause a switch from the physiological state to enhanced bone resorption or formation. This study investigated the mRNA expression of RANKL and osteoprotegerin, as well as their relative ratio, in the gingival tissues of patients with various forms of periodontal diseases. Gingival tissue was obtained from nine healthy subjects and 41 patients, who had gingivitis, chronic periodontitis, generalized aggressive periodontitis, and chronic periodontitis and were receiving immunosuppressant therapy. Quantitative real-time polymerase chain reaction was employed to evaluate the mRNA expression of RANKL and osteoprotegerin in these tissues. Compared with healthy individuals, patients in all periodontitis groups, but not those with gingivitis, exhibited stronger RANKL expression and a higher relative RANKL/osteoprotegerin ratio. In addition, osteoprotegerin expression was weaker in patients with chronic periodontitis. When patients with generalized aggressive periodontitis and chronic periodontitis were compared, the former exhibited stronger RANKL expression, whereas the latter exhibited weaker osteoprotegerin expression, and there was no difference in their relative ratio. When chronic periodontitis patients were compared with chronic periodontitis patients receiving immunosuppressant therapy, osteoprotegerin, but not RANKL, expression was stronger in the latter. This study demonstrates that RANKL and osteoprotegerin expression are differentially regulated in various forms of periodontitis, and the relative RANKL/osteoprotegerin ratio appears to be indicative of disease occurrence. This information may confer diagnostic and therapeutic value in periodontitis.

Differences in osteoclast formation between proximal and distal tibial osteoporosis in rats with adjuvant arthritis: inhibitory effects of bisphosphonates on osteoclasts

Patients with rheumatoid arthritis commonly suffer both systemic and periarticular osteoporosis. Bisphosphonates (BPs) are inhibitors of bone resorption, and several derivatives have been developed for treatment of enhanced bone resorption. We aimed to characterize osteoclast formation in two different sites, the proximal tibial and distal tibial areas, in rats with adjuvant arthritis, and to investigate the impact of amino or non-amino types of bisphosphonate. Adjuvant arthritis was initiated in rats while administering daily injections of either etidronate, a non-amino BP, or alendronate, an amino BP, for 3 weeks. On the day following the last injection, bone mineral density (BMD) was measured in the proximal tibia to assess systemic osteoporosis and in the distal tibia for periarticular osteoporosis using dual-energy X-ray absorptiometry. Subsequently, bone marrow cells from either end of the tibia were collected and incubated for 7 days before staining and counting tartrate-resistant acid phosphatase positive cells. In the rats with adjuvant arthritis, BMD of either end of the tibia was lower than in normal rats. Although etidronate prevented bone mineral loss at both ends, distal loss was significantly less than proximal. In contrast, alendronate significantly inhibited mineral loss primarily in the proximal area. Large osteoclasts, defined as having five or more nuclei, formed preferentially in the proximal tibia, while small osteoclasts with fewer than four nuclei were found mainly distally. The suppressive effect of alendronate was greater on the large osteoclasts, while etidronate had a greater effect on the small osteoclasts. These results show that the size and multinuclearity of osteoclasts and the number of osteoclasts formed are different in the distal and proximal areas of the tibia, and that alendronate and etidronate may suppress different types of osteoclasts as discriminated by the number of nuclei.

Differences in osteoclast formation between proximal and distal tibial osteoporosis in rats with adjuvant arthritis: inhibitory effects of bisphosphonates on osteoclasts

Patients with rheumatoid arthritis commonly suffer both systemic and periarticular osteoporosis. Bisphosphonates (BPs) are inhibitors of bone resorption, and several derivatives have been developed for treatment of enhanced bone resorption. We aimed to characterize osteoclast formation in two different sites, the proximal tibial and distal tibial areas, in rats with adjuvant arthritis, and to investigate the impact of amino or non-amino types of bisphosphonate. Adjuvant arthritis was initiated in rats while administering daily injections of either etidronate, a non-amino BP, or alendronate, an amino BP, for 3 weeks. On the day following the last injection, bone mineral density (BMD) was measured in the proximal tibia to assess systemic osteoporosis and in the distal tibia for periarticular osteoporosis using dual-energy X-ray absorptiometry. Subsequently, bone marrow cells from either end of the tibia were collected and incubated for 7 days before staining and counting tartrate-resistant acid phosphatase positive cells. In the rats with adjuvant arthritis, BMD of either end of the tibia was lower than in normal rats. Although etidronate prevented bone mineral loss at both ends, distal loss was significantly less than proximal. In contrast, alendronate significantly inhibited mineral loss primarily in the proximal area. Large osteoclasts, defined as having five or more nuclei, formed preferentially in the proximal tibia, while small osteoclasts with fewer than four nuclei were found mainly distally. The suppressive effect of alendronate was greater on the large osteoclasts, while etidronate had a greater effect on the small osteoclasts. These results show that the size and multinuclearity of osteoclasts and the number of osteoclasts formed are different in the distal and proximal areas of the tibia, and that alendronate and etidronate may suppress different types of osteoclasts as discriminated by the number of nuclei.

SB431542, a TGF-Beta Receptor Kinase Inhibitor, Restores Bone Formation Which Ameliorates Myeloma-Induced Microenvironment.

Multiple myeloma (MM) cells stimulate bone resorption and concomitantly suppress bone formation, leading to devastating bone destruction. TGF-beta, a potent inhibitor for terminal osteoblasts (OB) maturation and mineralization, is abundantly produced and released from bone tissues through enhanced bone resorption, and activated by osteoclasts (OC)-derived acids and MMPs in MM bone lesions. In the present study we investigated the impact of TGF-beta inhibition on induction of bone formation in MM as well as the effects of matured OB on MM growth. TGF-beta completely suppressed BMP-2-induced mineralized nodule formation by OB cultured in osteogenic media supplemented with beta-glycerophosphate and vitamin C. SB431542, an inhibitor of TGF-beta type I receptor kinase, potently suppressed induction of Smad6 by TGF-beta, which inhibits BMP-2 signaling. SB431542 along with BMP-2 abolished such TGF-beta actions and enhanced mineralized nodule formation more than BMP-2 alone. Notably, addition of SB431542 antagonized the inhibitory effects of conditioned media from MM cell lines (RPMI8226 and U266) and bone marrow plasma from patients with MM, resulting in restoration of the BMP-induced mineralized nodule formation. Furthermore, MC3T3-E1 cells matured by BMP-2 enough to exhibit mineralized nodules suppressed the proliferation of 5TGM1 MM cells in sharp contrast to stromal cells as well as untreated or TGF-beta-treated undifferentiated MC3T3-E1 cells that promote MM cell growth and survival. Interestingly, 5TGM1 MM cell growth was also potently suppressed by MC3T3-E1 cells cultured with BMP-2 in the absence of beta-glycerophosphate to achieve OB maturation without formation of mineralized nodules, suggesting a responsible role for OB-derived non-mineralized factors associated with terminal differentiation of OB. In addition, the induction of OB maturation down-regulated the production by OB of IL-6 and up-regulated osteoprotegerin, an inhibitor for osteoclastogenesis. Taken together, blockade of TGF-beta actions release OB from the differentiational arrest in MM bone disease and thus can be a good therapeutic maneuver restoring bone formation as well as suppressing osteoclastogenesis to ameliorate bone destruction and at the same time suppressing MM expansion by disrupting the MM-induced microenvironment which can be called as MM niche.