Receptor tyrosine kinase inhibition causes simultaneous bone loss and excess bone formation within growing bone in rats.

During postnatal skeletal growth, adaptation to mechanical loading leads to cellular activities at the growth plate. It has recently become evident that bone forming and bone resorbing cells are affected by the receptor tyrosine kinase (RTK) inhibitor imatinib mesylate (STI571, Gleevec®). Imatinib targets PDGF, ABL-related gene, c-Abl, c-Kit and c-Fms receptors, many of which have multiple functions in the bone microenvironment. We therefore studied the effects of imatinib in growing bone. Young rats were exposed to imatinib (150mg/kg on postnatal days 5-7, or 100mg/kg on postnatal days 5-13), and the effects of RTK inhibition on bone physiology were studied after 8 and 70days (3-day treatment), or after 14days (9-day treatment). X-ray imaging, computer tomography, histomorphometry, RNA analysis and immunohistochemistry were used to evaluate bone modeling and remodeling in vivo. Imatinib treatment eliminated osteoclasts from the metaphyseal osteochondral junction at 8 and 14days. This led to a resorption arrest at the growth plate, but also increased bone apposition by osteoblasts, thus resulting in local osteopetrosis at the osteochondral junction. The impaired bone remodelation observed on day 8 remained significant until adulthood. Within the same bone, increased osteoclast activity, leading to bone loss, was observed at distal bone trabeculae on days 8 and 14. Peripheral quantitative computer tomography (pQCT) and micro-CT analysis confirmed that, at the osteochondral junction, imatinib shifted the balance from bone resorption towards bone formation, thereby altering bone modeling. At distal trabecular bone, in turn, the balance was turned towards bone resorption, leading to bone loss.

A rationale for osteoclast selectivity of inhibiting the lysosomal V-ATPase a3 isoform.

Osteoclastic bone resorption can be completely abolished by inhibiting the vacuolar H(+)-ATPase (V-ATPase), a proton pump composed of at least 12 different subunits. However, V-ATPases are ubiquitous and it is unclear whether the osteoclast V-ATPase has a unique composition that would allow its selective inhibition. Aiming to answer this question, we compared human osteoclasts and monocytic THP.1 cells with respect to the localization of the a3 isoform of the 116-kDa subunit, which is indispensable for bone resorption, and sensitivity to SB242784, a V-ATPase inhibitor that prevents experimentally induced osteoporosis. By immunofluorescence, a3 was essentially nondetectable in THP.1 cells, while in osteoclasts a3 was highly upregulated and localized to lysosomes in nonresorbing osteoclasts. We isolated the lysosomal compartment from both sources as latex bead-containing phagolysosomes and compared them. Osteoclast phagolysosomes and THP.1 phagolysosomes both contained a3 and a1; however, the a3/a1 ratio was 3.8- to 11.2-fold higher in osteoclast phagolysosomes. Importantly, the V-ATPase-dependent acidification of phagolysosomes from both sources was essentially equally sensitive to SB242784. Thus, we observed no indication of a qualitative uniqueness of the osteoclast V-ATPase; rather, the high a3-level in osteoclasts may represent an upregulation of the common lysosomal V-ATPase. Our results, together with the reported phenotype of a3 deficiency and the reported efficacy of SB242784 in vivo, suggest that V-ATPase structure-independent mechanisms render bone resorption more sensitive than lysosomal function to V-ATPase inhibition. One such mechanism may be compensation of a3 by a1, which may be sufficient for retaining lysosomal function but not bone resorption.

FGF-8b induces growth and rich vascularization in an orthotopic PC-3 model of prostate cancer.

Fibroblast growth factor 8 (FGF-8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF-8b on proliferation of PC-3 prostate cancer cells and growth of PC-3 tumors, and to identify FGF-8b-associated molecular targets. Expression of ectopic FGF-8b in PC-3 cells caused a 1.5-fold increase in cell proliferation in vitro and a four- to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF-8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT-qPCR of FGF-8b mRNA. Microarray analyses revealed significantly altered, up- and downregulated, genes in PC-3 cell cultures (169 genes) and in orthotopic PC-3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell-to-cell signaling and energy production, and the downregulated genes associated with differentiation (DKK-1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT-qPCR. In conclusion, our results demonstrate that FGF-8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF-8b actions on prostate cancer progression and metastasis.

Overexpression of vascular endothelial growth factor C increases growth and alters the metastatic pattern of orthotopic PC-3 prostate tumors.

BACKGROUND: Prostate cancer metastasizes to regional lymph nodes and distant sites but the roles of lymphatic and hematogenous pathways in metastasis are not fully understood. METHODS: We studied the roles of VEGF-C and VEGFR3 in prostate cancer metastasis by blocking VEGFR3 using intravenous adenovirus-delivered VEGFR3-Ig fusion protein (VEGFR3-Ig) and by ectopic expression of VEGF-C in PC-3 prostate tumors in nude mice. RESULTS: VEGFR3-Ig decreased the density of lymphatic capillaries in orthotopic PC-3 tumors (p < 0.05) and inhibited metastasis to iliac and sacral lymph nodes. In addition, tumor volumes were smaller in the VEGFR3-Ig-treated group compared with the control group (p < 0.05). Transfection of PC-3 cells with the VEGF-C gene led to a high level of 29/31 kD VEGF-C expression in PC-3 cells. The size of orthotopic and subcutaneous PC-3/VEGF-C tumors was significantly greater than that of PC-3/mock tumors (both p < 0.001). Interestingly, while most orthotopic PC-3 and PC-3/mock tumors grown for 4 weeks metastasized to prostate-draining lymph nodes, orthotopic PC-3/VEGF-C tumors primarily metastasized to the lungs. PC-3/VEGF-C tumors showed highly angiogenic morphology with an increased density of blood capillaries compared with PC-3/mock tumors (p < 0.001). CONCLUSION: The data suggest that even though VEGF-C/VEGFR3 pathway is primarily required for lymphangiogenesis and lymphatic metastasis, an increased level of VEGF-C can also stimulate angiogenesis, which is associated with growth of orthotopic prostate tumors and a switch from a primary pattern of lymph node metastasis to an increased proportion of metastases at distant sites.

Evidence for the role of osteocytes in the initiation of targeted remodeling.

Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Osteocytes are the most abundant cells in bone, and their death by microdamage has been suggested to be the major event leading in the initiation of osteoclastic bone resorption. Even though there is increasing evidence that osteocyte density, microcracks and targeted remodeling are related, there still exist several questions. For example, how osteoclasts are targeted to the specific site of microdamage for repair. It has been proposed that apoptotic osteocytes could secrete a specific signal to target osteoclasts. The other question is the nature of this signal. To elucidate the role of microdamage-induced osteocyte cell death in the initiation of targeted remodelling, this paper discusses the potential use of an in vitro model, in which osteocytes can be three-dimensionally cultured and locally damaged. Furthermore, the method enables one to study the osteocyte-derived soluble interactions with bone marrow cells. It was demonstrated that damaged osteocytes locally affect osteoclast precursors by secreting osteoclastogenic factors, and thus can have a role in the initiation of resorption in bone remodelling. This strongly supports the idea that damage to osteocyte cellular network has the potential to stimulate osteoclastic proliferation and therefore the activation of Basic Multicellular Units (BMUs).

Serial assessment of serum bone metabolism markers identifies women with the highest rate of bone loss and osteoporosis risk.

CONTEXT: One of the important challenges in the management of osteoporosis is to identify women who are at high risk of developing osteoporosis and fragility fractures. OBJECTIVE: Our objective was to evaluate whether assessment of bone metabolism at multiple occasions can identify women with the highest risk for bone loss. DESIGN: The Malmö Osteoporosis Prospective Risk Assessment study is an ongoing longitudinal study. Participants have been evaluated at baseline and after 1, 3, and 5 yr. SETTING: We conducted a population-based study. PARTICIPANTS: Participants included 1044 women, all 75 yr old at baseline. MAIN OUTCOME MEASURES: Seven bone turnover markers were assessed at baseline and at 1, 3, and 5 yr (n = 573). The 5-yr change in areal bone mineral density (aBMD) was also determined. RESULTS: Baseline markers correlated weakly to change in total body aBMD. The associations were more pronounced when the average of the baseline and 1-yr measurements was used (standardized regression coefficients -0.12 to -0.23, P < 0.01). Adding the 3-yr and 5-yr measurement further strengthened the correlation (regression coefficients up to -0.30, P < 0.001). Women with constantly high turnover lost significantly more bone at total body assessment (-2.6%) than women with intermediate (-1.6%) or low turnover (-0.2%, P for trend < 0.001). They also had a greater decrease in hip BMD (-8.3, -6.0, and -5.1%, respectively, P = 0.010). Results were similar also in the subgroup of women with osteopenia. CONCLUSIONS: Our results suggest that serial assessment of bone turnover improves the identification of women with the highest rate of bone loss and osteoporosis risk.

FGF-8 is involved in bone metastasis of prostate cancer.

Prostate cancer is the most common malignancy of men in Western countries. Patients with advanced prostate cancer suffer from incurable bone metastases. Recent data indicate that interactions between prostate cancer cells, osteoblasts, osteoclasts and the bone matrix are essential in the formation of bone metastases. FGF-8 is widely overexpressed in prostate cancer. Recently, FGF-8 has been found to affect both osteoblast and osteoclast differentiation. The aim of this study was to examine the role of FGF-8 in bone metastasis of prostate cancer. Immunohistochemistry was used to analyse FGF-8 expression in clinical samples of prostate cancer bone metastases. The functional significance of FGF-8 in growth of bone metastasis and formation of bone lesions was verified by using intratibial inoculations of FGF-8 or mock transfected PC-3 prostate cancer cells in nude mice. Intratibial tumors and bone lesions were analysed with X-ray, micro-CT and detailed histomorphometry using image analysis software and with immunostaining for osteocalcin and cathepsin K. Immunohistochemical analysis of tissue microarray of bone metastases of human prostate cancer showed that 76% of human bone metastasis samples (n = 25 from 11 patients) were positive for FGF-8. FGF-8 increased the growth of intratibial tumors and local formation of lytic and sclerotic lesions of bone. These results demonstrate that FGF-8 is expressed at a high frequency in bone metastases of human prostate cancer and that expression of FGF-8 in PC-3 prostate cancer cells increases their growth as intratibial tumors and modulates formation of bone lesions in an in vivo model of prostate cancer bone metastasis.

Estrogen and the selective estrogen receptor modulator (SERM) protection against cell death in estrogen receptor alpha and beta expressing U2OS cells.

In the current work, we compared the ability of 17beta-estradiol (E2) and the selective estrogen receptor modulators (SERMs), tamoxifen (Tam), raloxifene (Ral) and ospemifene (Osp) to promote the survival of osteoblast-derived cells against etoposide-induced apoptosis. In order to compare the roles of the two estrogen receptor (ER) isotypes, we created a U2OS human osteosarcoma cell line stably expressing either ERalpha (ERalpha) or ERbeta (ERbeta). Transfection with either of the ERs was able to render the U2OS cells sensitive to E2. We show that E2 opposed etoposide-induced apoptosis and that the effect was mediated via both ER isotypes. The ER isotype selective agonists propyl-pyrazole-triol (PPT) and diarylpropionitrile (DPN) had the same effect in U2OS/ERalpha and U2OS/ERbeta cells, respectively. Osp also opposed apoptosis at least in U2OS/ERalpha cells. Tam and Ral were not able to protect against etoposide-induced cell death. In order to evaluate the protective effects of E2 and Osp upon etoposide challenge, we studied the expression of two E2-regulated, osteoblast-produced cytokines, IL-6 and OPG in E2 and SERM-treated U2OS/ERalpha and U2OS/ERbeta cells. Etoposide strongly increased expression of IL-6 and decreased that of OPG. E2 opposed IL-6 increase only in U2OS/ERalpha cells and OPG decrease primarily in ERbeta cells. Osp opposed the effect of etoposide on OPG primarily in U2OS/ERbeta cells but interestingly, it had little effect on IL-6 expression. E2, PPT, DNP and Osp also inhibited etoposide-induced death and cytokine changes in SAOS-2 osteosarcoma cells expressing endogenous ERalpha and ERbeta. Collectively, our results suggest that the osteoblast protective anti-apoptotic effects of E2 are mediated by both ERalpha and ERbeta but those of Osp primarily by ERalpha. In addition, E2 and Osp opposed the etoposide-induced increase of IL-6 and decrease of OPG which changes would increase osteoclastic activity. These anti-resorptive effects of E2 and Osp upon etoposide challenge differed from each other and they seemed to be differentially mediated in ERalpha and ERbeta expressing osteoblast-derived U2OS cells.

Osteoclast lineage and function.

Osteoclasts are members of the monocyte/macrophage lineage and are formed by cellular fusions from their mononuclear precursors. Their differentiation is regulated by a number of other cells and their products, especially by RANKL and M-CSF. The resorbing osteoclasts are polarized and show specific plasma membrane domains. Polarization and bone resorption need a continuous membrane trafficking and modulation of the cytoskeleton. The most characteristic feature of osteoclasts is their unique capacity to dissolve crystalline hydroxyapatite by targeted secretion of HCl into the extracellular resorption lacuna. Organic matrix is degraded by enzymes like cathepsin K and the degradation products are transcytosed through the cell for secretion. Dissolution of hydroxyapatite releases large amounts of soluble calcium, phosphate and bicarbonate. Removal of these ions apparently involves the vesicular pathways and direct ion transport via different ion exchangers, channels and pumps. Detailed molecular knowledge of osteoclast differentiation and function has helped us to identify several target molecules and develop specific treatments to inhibit pathological bone resorption in various skeletal diseases.

Clinical performance of six different serum tartrate-resistant acid phosphatase assays for monitoring alendronate treatment.

Two forms of tartrate-resistant acid phosphatase (TRACP) circulate in human blood, TRACP 5a derived from inflammatory macrophages and TRACP 5b derived from osteoclasts. We compared the clinical performance of the following TRACP immunoassays for monitoring alendronate treatment in postmenopausal women: 1) TRACP 5b activity using a selective pH; 2) TRACP 5b activity using a selective substrate; 3) Total TRACP activity; 4) Total TRACP protein amount; 5) TRACP 5a activity; 6) TRACP 5a protein amount. TRACP and other bone turnover markers were measured before the start of treatment and at 3 months. Alendronate treatment decreased TRACP values determined with assays 1, 2 and 3, and had no effect on the values determined with assays 4, 5 and 6. Clinical performance of assays 1, 2 and 3 was good, and these assays correlated with each other and with the other bone markers. This study showed that TRACP 5b specific methods are useful for monitoring changes in bone resorption during alendronate treatment, and alendronate treatment does not affect serum TRACP 5a levels.

Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells.

Bisphosphonates are used for prevention of osteoporosis and metastatic bone diseases. Anti-invasive effects on various cancer cells have also been reported, but the mechanisms involved are not well-understood. We investigated the effects of the nitrogen-containing bisphosphonate alendronate (ALN) on the regulation of actin cytoskeleton in PC-3 cells. We analyzed the ALN effect on the organization and the dynamics of actin, and on the cytoskeleton-related regulatory proteins cofilin, p21-associated kinase 2 (PAK2), paxillin and focal adhesion kinase. Immunostainings of cofilin in ALN-treated PC-3 cells and xenografts were performed, and the role of cofilin in ALN-regulated F-actin organization and migration/invasion in PC-3 cells was analyzed using cofilin knockdown and transfection. We demonstrate that disrupted F-actin organization and decreased cell motility in ALN-treated PC-3 cells were associated with decreased levels of total and phosphorylated cofilin. PAK2 levels were also lowered but adhesion-related proteins were not altered. The knockdown of cofilin similarly impaired F-actin organization and decreased invasion of PC-3 cells, whereas in the cells transfected with a cofilin expressing vector, ALN treatment did not decrease cellular cofilin levels and migration as in mock transfected cells. ALN also reduced immunohistochemical staining of cofilin in PC-3 xenografts. Our results suggest that reduction of cofilin has an important role in ALN-induced disruption of the actin cytoskeleton and inhibition of the PC-3 cell motility and invasion. These data also support the idea that the nitrogen-containing bisphosphonates could be efficacious in inhibition of prostate cancer invasion and metastasis, if delivered in a pharmacological formulation accessible to the tumors.

Associations between homocysteine, bone turnover, BMD, mortality, and fracture risk in elderly women.

UNLABELLED: Homocysteine has been suggested to be a risk factor for fracture, but the causal relationship is not clear. In 996 women from the OPRA study, high homocysteine level was associated with high bone marker levels and low BMD at baseline. During a mean 7-year follow-up, high homocysteine level was associated with mortality, but no clear association to fracture risk existed. INTRODUCTION: Recently, the association between high serum homocysteine (Hcy) levels and an increased risk of fracture has been described. MATERIALS AND METHODS: Hcy levels were measured at baseline in 996 women, all 75 years old. Vitamin B(12), folate, serum cross-linking telopeptide of type I collagen (CTX), serum TRACP5b, serum osteocalcin, urine deoxypyridinoline, PTH, areal BMD (aBMD), calcaneal quantitative ultrasound (QUS), and physical performance were assessed at baseline. Fractures and mortality were recorded during a mean follow-up of 7.0 years. RESULTS: Bone marker levels were higher in women with Hcy in the highest quartile compared with all other women (p < 0.05). The most evident correlation between Hcy and a bone marker was seen with CTX (r = 0.19, p < 0.001). aBMD (hip) was 4% lower, QUS was up to 2% lower, and gait speed was 11% slower among women with Hcy in the highest quartile compared with the other women (p < 0.05). During the follow-up, 267 women sustained at least one low-energy fracture (including 69 hip fractures). When women in the highest Hcy quartile were compared with all other women, the hazard ratios (HRs) for sustaining any type of fracture was 1.18 (95% CI, 0.89-1.36) and for hip fracture was 1.50 (95% CI, 0.91-1.94). For the same group of women, the mortality risk was 2.16 (95% CI, 1.58-2.55). Adjustments for confounders did not substantially change these associations. Adjustment for PTH increased the HR for hip fracture to 1.67 (95% CI, 1.01-2.17). Low vitamin B(12) or folate was not associated with increased fracture risk or mortality. CONCLUSIONS: High Hcy levels were associated with higher bone turnover, poor physical performance, and lower BMD. There was no clear association to fracture risk. The increased mortality among women with high Hcy levels indicates that a high Hcy level may be a marker of frailty.

Membrane-bound carbonic anhydrases in osteoclasts.

Osteoclasts are multinucleated bone-resorbing cells that use multiple pH regulation mechanisms to create an acidic pH in the resorption lacuna. Carbonic anhydrase II and vacuolar H(+)-ATPases produce and transport protons, while chloride channels provide a Cl(-) flux into the resorption site. These activities are required for inorganic matrix dissolution that precedes enzymatic removal of organic bone matrix. In other cell types it has become evident that carbonic anhydrase isoenzymes interact with AE proteins to form transport metabolons that regulate intracellular pH. Membrane-bound carbonic anhydrase isoenzymes may also compensate for the lack of cytoplasmic carbonic anhydrase II. Therefore, our goal was to explore the expression of membrane-bound carbonic anhydrase (CA) isoenzymes CA IV, CA IX, CA XII and CA XIV in bone-resorbing osteoclasts. Immunohistochemistry and confocal microscopy showed expression of CA IV, CA XII and CA XIV in cultured rat and human osteoclasts. To confirm these results, RT-PCR was used. Immunohistochemistry revealed distinct staining patterns for CA IV, CA XII and CA XIV in rat trabecular bone specimens. A plasma membrane staining was observed in bone lining cells with the CA XII antibody while osteoclast plasma membranes were stained with CA IV and CA XIV antibodies. Confocal microscopy of cultured human osteoclasts showed a punctated intracellular CA IV staining and a perinuclear CA XIV staining while no CA IX or CA XII staining was observed. To evaluate the physiological role of membrane-bound CAs in osteoclasts, we used PCS, a novel membrane-impermeable CA inhibitor. Increased osteoclast number and bone resorption activity was observed in rat osteoclast cultures exposed to a low concentration of PCS while higher concentrations affected cell survival. PCS treatment also disturbed intracellular acidification in osteoclasts, as determined by live cell microscopy. In conclusion, our data shows that membrane-bound carbonic anhydrase isoenzymes CA IV and CA XIV are expressed both at mRNA and protein levels in osteoclasts in vivo and in vitro. In addition, the inhibitor experiments provide novel evidence to support the hypothesis that intracellular pH regulation in osteoclasts may indeed involve transport metabolons.

RNA interference tolerates 2'-fluoro modifications at the Argonaute2 cleavage site.

Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2'-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2'-fluorinated 2'-deoxypurines and/or 2'-deoxypyrimidines in the antisense and/or sense strands. The capacity of 2'-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2'-F-modified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2'-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.

Pharmacological sequestration of intracellular cholesterol in late endosomes disrupts ruffled border formation in osteoclasts.

UNLABELLED: We showed that the ruffled border lacks a late endosomal lipid, LBPA, but is enriched incholesterol. A hydrophobic amine, U18666A, causes cholesterol accumulation in LBPA+ late endosomes in osteoclasts. Specific targeting of cathepsin K and the vacuolar H+-ATPase at the ruffled border is blocked by U18666A. A membrane trafficking pathway from baso-lateral membrane toward the resorptive organelle is also arrested by the inhibitor. These results indicate cholesterol homeostasis regulates late endosomal/lysosomal trafficking and polarized secretion in resorbing osteoclasts. INTRODUCTION: Protons and acidic proteases are secreted into the resorption lacuna through the ruffled border to solubilize bone mineral and digest the organic bone matrix, respectively. Whereas evidence suggests this event occurs through a vesicular trafficking mechanism, this issue remains unresolved. MATERIALS AND METHODS: The distribution of lysobisphosphatidic acid (LBPA) and cholesterol in resorbing osteoclasts was examined by laser scanning confocal microscopy. The effects of U18666A on ruffled border formation were observed by electron microscopy. RESULTS AND CONCLUSIONS: The ruffled border does not contain LBPA but is enriched in cholesterol. We found a hydrophobic amine, U18666A, which blocks the efflux of cholesterol from late endosomes in other cells, causes cholesterol accumulation in LBPA-containing late endosomes in osteoclasts, leading to diminished cholesterol at the ruffled border. Reflecting the U18666A-mediated inhibition of late endosome/lysosome transport, the resorptive membrane is disrupted and contains a paucity of cathepsin K and the vacuolar H+-ATPase. These results indicate that the ruffled border is formed by the fusion of lysosomes with the plasma membrane in osteoclasts through a process that is cholesterol regulated.

Bone marrow cell differentiation induced by mechanically damaged osteocytes in 3D gel-embedded culture.

UNLABELLED: Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover after microdamage. To study the role of osteocytes in targeted remodeling, we developed an in vitro model, in which osteocytes can be locally damaged and their interactions with bone marrow cells studied. Our results show that the damaged osteocytes activate the osteoclast precursors by soluble factors and thus can control the initial phase of targeted remodeling. INTRODUCTION: Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Besides the targeted remodeling, some remodeling may also be random to serve metabolic purposes. Osteocytes have been considered to provide a crucial role in the activation of osteoclastic bone resorption adjacent to the damaged site. This study was aimed to develop a relevant in vitro model of the targeted remodeling and to show that damaged osteocytes can induce the initial bone resorptive stage. MATERIALS AND METHODS: We developed a new device, in which osteocyte-like cell line MLO-Y4 cells were 3D cultured, subjected to local scratching, and assayed for cell viability. NIH3T3-3 cells were used as a control. Bone marrow cells were cultured on the top of the mechanically damaged MLO-Y4 cells, and the formation of TRACP+ cells was assayed. Additionally, the conditioned medium from scratched cultures was added to bone marrow cultures, and the TRACP activity in cell lysates was quantified. The macrophage-colony stimulating factor (M-CSF) and RANKL secretion in the conditioned medium was assayed by ELISA. RESULTS: Scratching induced the death of MLO-Y4 cells. When bone marrow cells were cultured over the gel-embedded MLO-Y4 cells, the application of mechanical scratching induced TRACP+ cell differentiation on gel surface. The cells with TRACP+ could be observed in the very restricted region along the scratching path. Additionally, mechanically damaged osteocytes secreted M-CSF and RANKL, and the conditioned medium showed the potential to induce TRACP+ cells in bone marrow culture. CONCLUSIONS: These findings indicate that soluble factors secreted from damaged osteocytes can locally induce and activate the initial phase of osteoclastic cell formation. This study directly shows the association between the damaged osteocytes and the initiation of resorptive stage in bone remodeling.

Regulation of osteoblast differentiation: a novel function for fibroblast growth factor 8.

Several members of the fibroblast growth factor (FGF) family have an important role in the development of skeletal tissues. FGF-8 is widely expressed in the developing skeleton, but its function there has remained unknown. We asked in this study whether FGF-8 could have a role in the differentiation of mesenchymal stem cells to an osteoblastic lineage. Addition of FGF-8 to mouse bone marrow cultures effectively increased initial cell proliferation as well as subsequent osteoblast-specific alkaline phosphatase production, bone nodule formation, and calcium accumulation if it was added to the cultures at an early stage of osteoblastic differentiation. Exogenous FGF-8 also stimulated the proliferation of MG63 osteosarcoma cells, which was blocked by a neutralizing antibody to FGF-8b. In addition, the heparin-binding growth factor fraction of Shionogi 115 (S115) mouse breast cancer cells, which express and secrete FGF-8 at a very high level, had an effect in bone marrow cultures similar to that of exogenous FGF-8. Interestingly, experimental nude mouse tumors of S115 cells present ectopic bone and cartilage formation as demonstrated by typical histology and expression of markers specific for cartilage (type II and IX collagen) and bone (osteocalcin). These results demonstrate that FGF-8 effectively predetermines bone marrow cells to differentiate to osteoblasts and increases bone formation in vitro. It is possible that FGF-8 also stimulates bone formation in vivo. The results suggest that FGF-8, which is expressed by a great proportion of malignant breast and prostate tumors, may, among other factors, also be involved in the formation of osteosclerotic bone metastases.

Cystatin B as an intracellular modulator of bone resorption.

Degradation of organic bone matrix requires proteinase activity. Cathepsin K is a major osteoclast proteinase needed for bone resorption, although osteoclasts also express a variety of other cysteine- and matrix metalloproteinases that are involved in bone remodellation. Cystatin B, an intracellular cysteine proteinase inhibitor, exhibits a lysosomal distribution preferentially in osteoclasts but it's role in osteoclast physiology has remained unknown. The current paper describes a novel regulatory function for cystatin B in bone-resorbing osteoclasts in vitro. Rat osteoclasts were cultured on bovine bone and spleen-derived cystatin B was added to the cultures. Nuclear morphology was evaluated and the number of actively resorbing osteoclasts and resorption pits was counted. Intracellular cathepsin K and tartrate-resistant acid phosphatase (TRACP) activities were monitored using fluorescent enzyme substrates and immunohistology was used to evaluate distribution of cystatin B in rat metaphyseal bone. Microscopical evaluation showed that cystatin B inactivated osteoclasts, thus resulting in impaired bone resorption. Cathepsin K and TRACP positive vesicles disappeared dose-dependently from the cystatin B-treated osteoclasts, indicating a decreased intracellular trafficking of bone degradation products. At the same time, cystatin B protected osteoclasts from experimentally induced apoptosis. These data show for the first time that, in addition to regulating cysteine proteinase activity and promoting cell survival in the nervous system, cystatin B inhibits bone resorption by down-regulating intracellular cathepsin K activity despite increased osteoclast survival.

Monocyte-macrophage system as a target for estrogen and selective estrogen receptor modulators.

Postmenopausal decline of estrogen production is associated with development of several degenerative disorders such as osteoporosis, neuroinflammatory diseases and vascular wall degeneration. These are associated with the activation of the cells of the monocyte-macrophage system in a context-dependent manner. Estrogen regulates differentiation, maturation and function of many cell types in this system directly or indirectly via other cells by autocrine/paracrine mechanisms. Estrogen effects on the monocyte-macrophage system are primarily repressive. Most of these effects are mediated by repression of expression of genes for cytokines or modulation of other inflammatory mediators by the estrogen receptor (ER)-dependent or nongenomic pathways. The ER-dependent mechanisms mostly involve modulation of the nuclear factor kappa B (NF-kappaB) pathway for transcriptional regulation of cytokine or other mediator genes. In the context of hormone-regulated cancer, estrogen can influence production of cytokines or other inflammatory mediators by both tumor cells and tumor-invading macrophages. The interactions of breast and prostate cancer cells with tumor-associated macrophages (TAMs) may play an important role in tumor progression and even in the development of resistance to hormonal treatment. Regulation of the monocyte-macrophage system by estrogen and cross-talk between the ER and cytokine-mediated pathways provides multiple novel targets for development of selective ER modulator (SERM) molecules for prevention and treatment of postmenopausal degenerative and neoplastic diseases.

Tartrate-resistant acid phosphatase 5b (TRACP 5b) as a marker of bone resorption.

Tartrate-resistant acid phosphatase (TRACP) is an enzyme that is expressed in high amounts by bone resorbing osteoclasts, inflammatory macrophages and dendritic cells. Two forms of TRACP circulate in human blood, TRACP 5a derived from macrophages and dendritic cells, and TRACP 5b derived from osteoclasts. Recent data have demonstrated the utility of TRACP 5b as a marker of osteoclast number and bone resorption, and serum TRACP 5a as a marker of inflammatory conditions. This review summarizes the scientific knowledge on the role of TRACP in osteoclastic bone resorption, the mechanism of TRACP 5b generation in osteoclasts and its secretion into the blood circulation, the methodology of measuring TRACP 5b, diagnostic evidence for the use of TRACP 5b as a resorption marker, and characteristics of TRACP 5b compared to other commonly used bone turnover markers.