High platelet count is associated with low bone mineral density: The MrOS Sweden cohort.

In elderly ambulatory men, high platelet and high neutrophil counts are related to low bone mineral density (BMD), after adjustment for relevant covariates. Low hemoglobin (hgb) is even associated with low BMD, but this relationship seems to be dependent on estradiol and osteocalcin. PURPOSE: Blood and bone cells exist in close proximity to each other in the bone marrow. Accumulating evidence, from both preclinical and clinical studies, indicates that these cell types are interconnected. Our hypothesis was that BMD measurements are associated with blood count variables and bone remodeling markers. METHODS: We analyzed blood count variables, bone remodeling markers, and BMD, in subjects from the MrOS cohort from Gothenburg, Sweden. Men with at least one blood count variable (hgb, white blood cell count, or platelet count) analyzed were included in the current analysis (n = 1005), median age 75.3 years (range 69-81 years). RESULTS: Our results show that high platelet counts were related to low BMD at all sites (total hip BMD; r = - 0.11, P = 0.003). No statistically significant association was seen between platelet counts and bone remodeling markers. Neutrophil counts were negatively associated with total body BMD (r = - 0.09, P = 0.006) and total hip BMD (r = - 0.08, P = 0.010), and positively related to serum ALP (r = 0.15, P < 0.001). Hgb was positively related to total hip BMD (r = 0.16, P < 0.001), and negatively to serum osteocalcin (r = - 0.13, P < 0.001). The association between platelet and neutrophil counts and total hip BMD was statistically significant after adjustments for other covariates, but the association between hgb and total hip BMD was dependent on estradiol and osteocalcin. CONCLUSIONS: Our observations support the hypothesis of an interplay between blood and bone components.

THE CRUCIAL ROLE OF THE WNT SYSTEM IN BONE REMODELLING.

The maintenance of bone mass is critically dependent on the balance between bone formation by osteoblasts and bone resorption by osteoclasts, processes in which osteocytes play also an important role. The activities of these bone cells are regulated by a variety of endocrine and paracrine factors of which sex steroids, parathyroid hormone, 1.25(OH)2-vitamin D3, glucocorticoids, retinoids and thyroid hormones are the most well known systemic factors. To the long list of locally acting factors belong cytokines and growth factors. This list was extended some 15 years ago by the discovery of the very important role of the WNT signalling system for the maintenance of bone mass. The first evidence of its role was the findings that mutations in the LRP5 gene, encoding a co-receptor in WNT-signaling, could result in either gain or loss of bone mass, i.e. either high bone mass or osteoporosis. This was a most unexpected observation since no indications existed prior to this discovery that the WNT signalling system had a role in bone remodeling. Since then, many observations have been made demonstrating the important role of different WNTs in regulating bone formation and resorption. Interestingly, some of these findings have demonstrated that trabecular and cortical bone are regulated by different mechanisms. It is the aim of the present overview to give the readers an insight into the WNT signalling system and its role in bone remodeling.

High plasma osteocalcin is associated with low blood haemoglobin in elderly men: the MrOS Sweden Study.

BACKGROUND: It has been suggested that osteoblasts are involved in the regulation of haematopoietic stem cells. Whether osteocalcin, which is derived from osteoblasts and is metabolically active, influences blood haemoglobin (Hb) levels is not known. OBJECTIVE: To determine whether plasma osteocalcin is a determinant of Hb in elderly men. METHODS: A total of 993 men (mean age 75.3 ± 3.2 years) participated in the population-based MrOS (osteoporotic fractures in men) study. Plasma osteocalcin concentration was evaluated in relation to Hb and adjustments were made for potential confounders (i.e. age, body mass index, erythropoietin, total oestradiol, fasting insulin, adiponectin, ferritin and cystatin C). RESULTS: Hb correlated (age adjusted) negatively with osteocalcin in the total study group (r = -0.12, P < 0.001) as well as in the subgroup of nondiabetic men (r = -0.16, P < 0.001). In nondiabetic men with higher osteocalcin levels, it was more likely that Hb would be in the lowest quartile (odds ratio per SD decrease in osteocalcin 1.32, 95% confidence interval 1.13-1.53). Quartiles of Hb were negatively associated (age adjusted) with osteocalcin (P < 0.001). Anaemic men (47/812) (Hb <130 g L(-1) ) had significantly higher mean osteocalcin levels than nonanaemic men (33.9 vs. 27.1 µg L(-1) , P < 0.001). In multiple stepwise linear regression analyses (adjusted for age, body mass index, total oestradiol, adiponectin, erythropoietin, fasting insulin, cystatin C, leptin, ferritin and holotranscobalamin), osteocalcin was an independent predictor of Hb concentration in nondiabetic men (P < 0.05). CONCLUSIONS: These data add further support to the evidence indicating that the bone-specific protein osteocalcin has several endocrine functions targeting the pancreas, testes, adipocytes, brain. An additional novel finding is that osteocalcin may also have a paracrine function as a regulator of haematopoiesis.

The WNT system: background and its role in bone.

WNTs are extracellular proteins that activate different cell surface receptors linked to canonical and noncanonical WNT signalling pathways. The Wnt genes were originally discovered as important for embryonic development of fruit flies and malignant transformation of mouse mammary cancers. More recently, WNTs have been implicated in a wide spectrum of biological phenomena and diseases. During the last decade, several lines of clinical and preclinical evidence have indicated that WNT signalling is critical for trabecular and cortical bone mass, and this pathway is currently an attractive target for drug development. Based on detailed knowledge of the different WNT signalling pathways, it appears that it might be possible to develop drugs that specifically target cortical and trabecular bone. Neutralization of a bone-specific WNT inhibitor is now being evaluated as a promising anabolic treatment for patients with osteoporosis. Here, we provide the historical background to the discoveries of WNTs, describe the different WNT signalling pathways and summarize the current understanding of how these proteins regulate bone mass by affecting bone formation and resorption.

High serum adiponectin is associated with low blood haemoglobin in elderly men: the Swedish MrOS study.

OBJECTIVES: Blood haemoglobin (Hb) concentration declines in elderly men, whilst the level of the adipocyte-derived protein adiponectin increases with age. The association between erythropoiesis and adiponectin in elderly men is unclear. The aim of this study was to determine whether adipokines such as adiponectin and leptin are associated with anaemia and Hb concentration in elderly community-dwelling men. DESIGN AND SETTING: The Gothenburg part of the population-based Swedish Osteoporotic Fractures in Men (MrOS) cohort (n = 1010; median age 75.3 years, range 69-81). MAIN OUTCOME MEASURES: We investigated the associations between levels of adiponectin and Hb before and after adjusting for potential confounders [i.e. age, body composition, erythropoietin (EPO), total oestradiol, leptin, cystatin C and iron and B vitamin status]. RESULTS: In these elderly men, age was negatively associated with Hb (r = -0.12, P < 0.001) and positively associated with adiponectin level (r = 0.13, P < 0.001). In age-adjusted partial correlations, Hb and adiponectin levels were negatively correlated (r = -0.20, P < 0.001); this association remained significant after multivariable adjustment for age, body composition, EPO, fasting insulin, sex hormones, leptin and ferritin. Age-adjusted mean adiponectin concentrations were significantly higher in anaemic men (66/1005; Hb <130 g L(-1) ) compared to nonanaemic men (14.0 vs. 11.7 µg mL(-1) , P < 0.05). In multivariate analysis, adiponectin together with EPO, total oestradiol, insulin, albumin, transferrin saturation, HDL cholesterol, cystatin C, total body fat mass and free thyroxine, but not leptin, explained 35% of the variation in Hb level. These results remained essentially unchanged after exclusion of men with diabetes. CONCLUSIONS: Serum adiponectin, but not leptin, was negatively and independently associated with Hb. This finding suggests a possible role of adiponectin in the age-related decline in Hb level observed in apparently healthy elderly men.

Low holotranscobalamin and cobalamins predict incident fractures in elderly men: the MrOS Sweden.

UNLABELLED: In a population-based study on cobalamin status and incident fractures in elderly men (n = 790) with an average follow-up of 5.9 years, we found that low levels of metabolically active and total cobalamins predict incident fractures, independently of body mass index (BMI), bone mineral density (BMD), plasma total homocysteine (tHcy), and cystatin C. INTRODUCTION: Cobalamin deficiency in elderlies may affect bone metabolism. This study aims to determine whether serum cobalamins or holotranscobalamin (holoTC; the metabolic active cobalamin) predict incident fractures in old men. METHODS: Men participating in the Gothenburg part of the population-based Osteoporotic Fractures in Men (MrOS) Sweden cohort and without ongoing vitamin B medication were included in the present study (n = 790; age range, 70-81 years). RESULTS: During an average follow-up of 5.9 years, 110 men sustained X-ray-verified fractures including 45 men with clinical vertebral fractures. The risk of fracture (adjusted for age, smoking, BMI, BMD, falls, prevalent fracture, tHcy, cystatin C, 25-OH-vitamin D, intake of calcium, and physical activity (fully adjusted)), increased per each standard deviation decrease in cobalamins (hazard ratio (HR), 1.38; 95% confidence intervals (CI), 1.11-1.72) and holoTC (HR, 1.26; 95% CI, 1.03-1.54), respectively. Men in the lowest quartile of cobalamins and holoTC (fully adjusted) had an increased risk of all fracture (cobalamins, HR = 1.67 (95% CI, 1.06-2.62); holoTC, HR = 1.74 (95% CI, 1.12-2.69)) compared with quartiles 2-4. No associations between folate or tHcy and incident fractures were seen. CONCLUSIONS: We present novel data showing that low levels of holoTC and cobalamins predicting incident fracture in elderly men. This association remained after adjustment for BMI, BMD, tHcy, and cystatin C. However, any causal relationship between low cobalamin status and fractures should be explored in a prospective treatment study.

Phytocystatin CsinCPI-2 Reduces Osteoclastogenesis and Alveolar Bone Loss.

Periodontal disease (PD) is a polymicrobial chronic inflammatory condition of the supporting tissues around the teeth, leading to the destruction of surrounding connective tissue. During the progression of PD, osteoclasts play a crucial role in the resorption of alveolar bone that eventually leads to the loss of teeth if the PD is left untreated. Therefore, the development of antiresorptive therapies targeting bone-resorbing cells will significantly benefit the treatment of PD. Here, we demonstrate the inhibitory effect of CsinCPI-2, a novel cysteine peptidase inhibitor from the orange tree, on periodontitis-induced inflammation, alveolar bone loss, and osteoclast differentiation. Using the ligature-induced periodontitis model in mice, we show that treatment with CsinCPI-2 (0.8 µg/g of body weight) significantly reduced inflammatory cell infiltrate in the connective tissue and prevented the loss of alveolar bone mass (BV/TV) caused by PD, effects associated with diminished numbers of TRAP-positive multinucleated cells. Furthermore, CsinCPI-2 significantly downregulated the numbers of inflammatory cells expressing CD3, CD45, MAC387, and IL-1ß. In vitro, CsinCPI-2 inhibited RANKL-induced TRAP+ multinucleated osteoclast formation in mouse bone marrow macrophage cultures in a concentration-dependent manner. This effect was not due to cytotoxicity, as demonstrated by the MTT assay. CsinCPI-2 inhibited RANKL-induced mRNA expression of Acp5, Calcr, and Ctsk, as well as the RANKL-induced upregulation of Nfatc1, a crucial transcription factor for osteoclast differentiation. Based on our findings, CsinCPI-2 prevents bone loss induced by PD by controlling the inflammatory process and acting directly on osteoclastogenesis, suggesting an interesting potential for CsinCPI-2 in the strategy for PD treatment.

IL-1beta and TNF-alpha regulate IL-6-type cytokines in gingival fibroblasts.

UNLABELLED: Interleukin-6 (IL-6)-type cytokines are pleiotropic molecules capable of stimulating bone resorption and expressed by numerous cell types. In the present study, we tested the hypothesis that gingival fibroblasts may exert local osteotropic effects through production of IL-6 and related cytokines. IL-6-type cytokine expression and regulation by IL-1beta and tumor necrosis factor-alpha (TNF-alpha) were studied in fibroblasts from the non-inflamed gingiva of healthy individuals. Constitutive mRNA expression of IL-6, IL-11, and leukemia inhibitory factor (LIF), but not of oncostatin M (OSM), was demonstrated, as was concentration-dependent stimulation of IL-6 and LIF mRNA and of protein by IL-1beta and TNF-alpha. IL-11 mRNA and protein were concentration-dependently stimulated by IL-1beta. The signaling pathway involved in IL-6 and LIF mRNA stimulation involved MAP kinases, but not NF-kappaB. The findings support the view that resident cells may influence the pathogenesis of periodontal disease through osteotropic IL-6-type cytokine production mediated by activation of MAP kinases. ABBREVIATIONS: IL-1alpha (interleukin-1alpha); IL-1beta (interleukin-1beta); IL-6 (interleukin-6); IL-11 (interleukin-11); LIF (leukemia inhibitory factor); OSM (oncostatin M); alpha(1)-coll. I (alpha(1)-collagen I); ALP (alkaline phosphatase); BMP-2 (bone morphogenetic protein-2); OC (osteocalcin); BSP (bone sialoprotein); TNFR I (tumor necrosis factor receptor I); TNFR II (tumor necrosis factor receptor II); IL-1R1 (interleukin-1 receptor 1); GAPDH (glyceraldehyde-3-phosphate dehydrogenase); RPL13A (ribosomal protein L13A); mRNA (messenger ribonucleic acid); cDNA (complementary deoxyribonucleic acid); PCR (polymerase chain-reaction); BCA (bicinchoninic acid); ELISA (enzyme-linked immunosorbent assay); alpha-MEM (alpha modification of Minimum Essential Medium); and FCS (fetal calf serum).

Osteotropic effects by the neuropeptides calcitonin gene-related peptide, substance P and vasoactive intestinal peptide.

Immunohistochemical phenotypic characterization of skeletal nerve fibers has demonstrated the expression of a restricted number of neuropeptides, including calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP). According to the neuro-osteological hypothesis, such neuropeptides can be released and exert paracrine biological effects on bone cells present close to the nerve endings expressing these signaling molecules. The existence of such interplay is most convincingly shown by the hypothalamic control of bone formation, in the case of leptin stimulation of hypothalamic nuclei mediated by the sympathetic nervous system and inhibitory beta-adrenergic receptors on osteoblasts. In addition to these receptors, osteoblasts and osteoclasts express functional receptors for CGRP, SP and VIP, which can regulate both bone formation and bone resorption. The evidence for these observations is summarized in the present paper.

In situ microdialysis in bone tissue. Stimulation of prostaglandin E2 release by weight-bearing mechanical loading.

In this study we describe, to our knowledge, the first experiment using the microdialysis technique for studying the release of prostaglandin E2 (PGE2) in the proximal tibia metaphysis secondary to mechanical loading. Nine healthy females, six in the loading group and three controls, mean age 34+/-2 (years+/-SEM), participated. A standard microdialysis catheter was inserted into the tibia metaphyseal bone under local anesthesia. Samplings were done every 15 min under a 2-h equilibration period. Thereafter, heel-drops (one impact per second) with as hard impact of the heels into the floor as possible, were done for 5 min by the subjects in the loading group. The control group performed no exercise. Sampling continued after this for another 2-h period. Basal levels of PGE2 in the proximal tibial metaphysis were determined to a mean of 45+/-10 pg/ml (mean+/-SEM, n = 6). The major finding was a 2.5-3.5-fold increase in the release of PGE2 after mechanical loading. The increase was statistically significant (P < 0.05 compared with basal levels) 1 h after the mechanical loading and persisted for the rest of the experimental period. No major alterations were observed in the control group. In conclusion, our data demonstrate that in situ microdialysis is a useful method for studying the PGE2 production in human bone. Furthermore, a rapid and significant increase in PGE2 levels was noticed in response to dynamic mechanical loading.

Bone remodeling in post-menopausal osteoporosis.

Bone mass in the skeleton is dependent on the coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts in discrete bone multi-cellular units. Remodeling of bone in these units is important not only for maintaining bone mass, but also to repair microdamage, to prevent accumulation of too much old bone, and for mineral homeostasis. The activities of osteoblasts and osteoclasts are controlled by a variety of hormones and cytokines, as well as by mechanical loading. Most importantly, sex hormones are very crucial for keeping bone mass in balance, and the lack of either estrogen or testosterone leads to decreased bone mass and increased risk for osteoporosis. The prevalence of osteoporotic fractures is increasing dramatically in the Western part of the world and is a major health problem in many countries. In the present review, the cellular and molecular mechanisms controlling bone remodeling and the influence of sex hormones on these processes are summarized. In a separate paper in this issue, the pathogenesis of post-menopausal osteoporosis will be compared with that of inflammation-induced bone remodeling, including the evidence for and against the hypothesis that concomitant post-menopausal osteoporotic disease influences the progression of periodontal disease.

Inflammation-induced bone remodeling in periodontal disease and the influence of post-menopausal osteoporosis.

During physiological conditions, the skeleton is remodeled in so-called bone multi-cellular units. Such units have been estimated to exist at 1-2 x 10(6) sites in the adult skeleton. The number and activities of these units are regulated by a variety of hormones and cytokines. In post-menopausal osteoporosis, lack of estrogen leads to increased numbers of bone multi-cellular units and to uncoupling of bone formation and bone resorption, resulting in too little bone laid down by osteoblasts compared with the amount of bone resorbed by osteoclasts. Inflammatory processes in the vicinity of the skeleton, e.g., marginal and apical periodontitis, will affect the remodeling of the nearby bone tissue in such a way that, in most patients, the amount of bone resorbed exceeds that being formed, resulting in net bone loss (inflammation-induced osteolysis). In some patients, however, inflammation-induced bone formation exceeds resorption, and a sclerotic lesion will develop. The cellular and molecular pathogenetic mechanisms in inflammation-induced osteolysis and sclerosis are discussed in the present review. The cytokines believed to be involved in inflammation-induced remodeling are very similar to those suggested to play crucial roles in post-menopausal osteoporosis. In patients with periodontal disease and concomitant post-menopausal osteoporosis, the possibility exists that the lack of estrogen influences the activities of bone cells and immune cells in such a way that the progression of alveolar bone loss will be enhanced. In the present paper, the evidence for and against this hypothesis is presented.

Deletions of genes encoding calcitonin/alpha-CGRP, amylin and calcitonin receptor have given new and unexpected insights into the function of calcitonin receptors and calcitonin receptor-like receptors in bone.

It has been suggested that skeletal nerves fibers may play important roles in neuro-osteogenic interactions. This view is partly based upon information obtained from immunohistochemical studies, chemical and surgical denervation experiments and clinical observations in patients with stroke and spinal cord injury, indicating the presence of a network of nerve fibers in the skeleton and that defective signalling in skeletal nerve fibers affects remodelling of bone. This view is also supported by data showing that functional receptors for signalling molecules in skeletal nerve fibers are expressed in bone cells and that activation of these receptors leads to profound effects on bone forming osteoblasts and bone resorbing osteoclasts. Convincing evidence for a role of neuronal signalling in bone metabolism has been provided by gene deletion approaches in which it has been shown that leptin-sensitive and neuropeptide Y-sensitive receptors in hypothalamus are important for bone remodelling in mice. Recently, gene deletion experiments have shown that calcitonin gene-related peptide (CGRP), one of the neuropeptides present in skeletal nerve fibers, is an important physiological regulator of bone formation at the level of osteoblast activity. CGRP belongs to the calcitonin (CT) family of peptides also including CT, amylin and adrenomedullin, as well as the recently described intermedin and calcitonin receptor-stimulating peptide. These peptides utilize two seven transmembrane G protein-coupled receptors - the calcitonin receptor (CTR) and the calcitonin receptor- like receptor (CRLR) - which can dimerize with three different single transmembrane proteins, making up the RAMP family. Associations between RAMPs and either CTR or CRLR give rise to seven distinct, molecularly characterized, receptors for CT, CGRP, amylin and adrenomedullin. Deletions of the genes for ligands in the CT family of peptides and for one of the receptors have revealed unexpected findings that have changed our view on the role of these peptides in bone remodelling. It was anticipated that deletions of the CT/alpha-CGRP and CTR genes would lead to bone loss, since CT has been shown to inhibit bone resorption in vitro and in vivo and has been used to treat patients with excessive bone resorption. Surprisingly, it was found that CT/alpha-CGRP-/- and CTR+/- mice have increased bone mass due to increased bone formation. Mice with deletion of the amylin gene, however, exhibited bone loss due to enhanced bone resorption. Selective deletion of the alpha-CGRP gene also leads to bone loss, but due to decreased bone formation. Thus, our understanding of the role of the CT family of peptides has been changed dramatically and much more data have to be gained before we fully understand the roles these peptides have in bone biology.

Inhibition of bone resorption in vitro by serine-esterase inhibitors.

The effect of two synthetic serine esterase inhibitors, N-alpha-dansyl(p-guanidino)phenylalaninepiperidine hydrochloride (I 2581) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (D-Phe-Pro-Arg-CH2Cl), on bone resorption in organ cultured mouse calvaria from neonatal mice has been examined. Mineral mobilization was assessed by analyzing the release of 45Ca, stable calcium (Ca2+) and inorganic phosphate (Pi). Organic matrix degradation was studied by analyzing the release of 3H from [3H]proline-labelled bones, and by quantifying the amounts of hydroxyproline in bone after culture. It was found that I 2581, at and above 30 mumol/l, dose-dependently inhibited 45Ca release induced by thrombin, parathyroid hormone (PTH), prostaglandin E2 and 1-alpha-hydroxyvitamin D-3. I 2581 (50 mumol/l) inhibited PTH-stimulated release of 3H from [3H]proline-labelled bones, and this effect was reversible after withdrawal of I 2581. I 2581 (50 mumol/l) inhibited the release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in bones stimulated by PTH and 1-alpha-hydroxyvitamin D-3, without affecting the release of lactate dehydrogenase. In parallel, I 2581 decreased PTH and 1-alpha-hydroxyvitamin D-3 induced reduction of hydroxyproline levels in bones after culture. I 2581 (50 mumol/l) did not affect the basal release of 45Ca, Ca2+, beta-glucuronidase and beta-N-acetylglucosaminidase, nor the basal amounts of hydroxyproline in bones after culture. D-Phe-Pro-Arg-CH2Cl (100 mumol/l) significantly inhibited PTH- and PGE2-induced release of 45Ca without affecting basal release of radioactive calcium. These data indicate that activation of serine proteinase(s) may be a necessary step in the mechanism of action of several stimulators of bone resorption.

Effects of cholera toxin on cyclic AMP accumulation and bone resorption in cultured mouse calvaria.

We have utilized the adenylate cyclase stimulator, cholera toxin, as a tool to test the role of cyclic AMP as a mediator of the effects on bone resorption by the calcium-regulating hormones, parathyroid hormone (PTH) and calcitonin. The effects on bone resorption were studied in an organ culture system using calvarial bones from newborn mice. Cyclic AMP response was assayed in calvarial bone explants and isolated osteoblasts from neonatal mouse calvaria. Cholera toxin caused a dose-dependent cAMP response in calvarial bones, seen at and above approx. 1-3 ng/ml and calculated half-maximal stimulation (EC50) at 18 ng/ml. The stimulatory effect of cholera toxin could be potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 0.2 mmol/l). Cyclic AMP accumulation in the bones was maximal after 4-6 h, and thereafter declined. However, activation of the adenylate cyclase was irreversible and the total amount (bone + medium) of cAMP produced, in the presence of IBMX (0.2 mmol/l), increased with time, for at least 48 h. In osteoblast-like cells cholera toxin (1 microgram/ml) stimulated the cellular levels of cAMP with a peak after 60-120 min, which could be potentiated with IBMX. The total cAMP accumulation indicated an irreversible response. In short-term bone organ cultures (at most, 24 h) cholera toxin, at and above 3 ng/ml, inhibited the stimulatory effect of PTH (10 nmol/l) on 45Ca release from prelabelled calvarial bones. The inhibitory effect of cholera toxin (0.1 microgram/ml) on 45Ca release was significant after 6 h and the calculated IC50 value at 24 h was 11.2 ng/ml. Cholera toxin (0.1 microgram/ml) also inhibited PTH-stimulated (10 nmol/l) release of Ca2+, inorganic phosphate (Pi), beta-glucuronidase, beta-N-acetylglucosaminidase and degradation of organic matrix (release of 3H from [3H]proline-labelled bones) in 24 h cultures. 45Ca release from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxyvitamin D3 (0.1 mumol/l) was also inhibited by cholera toxin (0.3 microgram/ml) in 24-h cultures. The inhibitory effect of cholera toxin on bone resorption was transient, and in long-term cultures (120 h) cholera toxin caused a dose-dependent, delayed stimulation of mineral mobilization (Ca2+, 45Ca, Pi), degradation of matrix and release of the lysosomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood coagulation and bone metabolism: some characteristics of the bone resorptive effect of thrombin in mouse calvarial bones in vitro.

Chronic inflammatory processes are often associated with bone resorption. Stimulated by the current great interest in the role of coagulation factors in inflammation and immune injury, we have studied the effect of thrombin on mouse calvarial bones in vitro. Thrombin caused a dose-dependent (0.1-7 U/ml) stimulation of 45Ca release from neonatal mouse calvarial bones. Thrombin also stimulated the mobilization of stable calcium and inorganic phosphate, the release of 3H from [3H]proline-labelled calvaria, the production of lactate and the release of the lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase. Thrombin also enhanced 45Ca release from fetal rat long bones, although this bone resorption assay was less sensitive to thrombin than the mouse calvarial system. The bone resorption stimulatory activity of thrombin in mouse calvaria could be inhibited by calcitonin and an increased concentration of phosphate in the culture medium. Thrombin-induced 45Ca release in mouse calvaria was sensitive to inhibition by hydrocortisone and dexamethasone. By contrast, 45Ca release response to parathyroid hormone was insensitive to corticosteroids. The prostaglandin synthetase inhibitors indomethacin, meclofenamic acid and naproxen and 5,8,11,14-eicosatetraynoic acid reduced 45Ca release from thrombin-stimulated calvaria. However, significant stimulation by thrombin could be achieved also in bones treated with inhibitors of arachidonate metabolism. The results obtained suggest that thrombin can stimulate cell-mediated bone resorption by an osteoclast-dependent mechanism. The mechanism of action may involve both prostaglandin-dependent and prostaglandin-independent pathways. Our findings indicate that thrombin may contribute to the bone resorptive processes seen in periodontal disease and rheumatoid arthritis.

Stimulation of resorption in cultured mouse calvarial bones by thiazolidinediones.

Dosage-dependent release of 45Ca was observed from prelabeled mouse calvarial bones after treatment with two thiazolidinediones, troglitazone and ciglitazone. Release of 45Ca by ciglitazone was decreased by the osteoclast inhibitors acetazolamide, calcitonin, 3-amino-1-hydroxypropylidene-1,1-bisphosphonate, and IL-4, but not affected by the peroxisome proliferator-activated receptor gamma antagonist, GW 9662, the mitotic inhibitor, hydroxyurea, or indomethacin. Enhanced expression of receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein and decreased osteoprotegerin (OPG) mRNA and protein were noted after ciglitazone treatment of calvariae. Ciglitazone and RANKL each caused increased mRNA expression of osteoclast markers: calcitonin receptor, tartrate-resistant acid phosphatase, cathepsin K, matrix metalloproteinase-9, integrin beta3, and nuclear factor of activated T cells 2. OPG inhibited mRNA expression of RANKL stimulated by ciglitazone, mRNA expression of osteoclast markers stimulated by ciglitazone and RANKL, and 45Ca release stimulated by troglitazone and ciglitazone. Increased expression of IL-1alpha mRNA by ciglitazone was not linked to resorption stimulated by the thiazolidinedione. Ciglitazone did not increase adipogenic gene expression but enhanced osteocalcin mRNA in calvariae. In addition to exhibiting sensitivity to OPG, data indicate that stimulation of osteoclast differentiation and activity by thiazolidinediones may occur by a nonperoxisome proliferator-activated receptor gamma-dependent pathway that does not require cell proliferation, prostaglandins, or IL-1alpha but is characterized by an increased RANKL to OPG ratio.

Modifications of the mouse calvarial technique improve the responsiveness to stimulators of bone resorption.

The effect of a preincubation period, in basic medium or in medium with inhibitors of prostaglandin biosynthesis, on the response to different stimulators of bone resorption has been studied in an organ culture system using calvarial bones from neonatal mice. Bone resorption was assessed either by the release of 45Ca or by the release of 3H from [3H]-proline labeled bones. Preincubated bones were cultured for 18-24 hr in medium, with and without indomethacin, hydrocortisone, and dexamethasone, and then extensively washed before being transferred to culture medium containing different stimulators of bone resorption. Preincubation in medium containing indomethacin or corticosteroids resulted in an increased response to parathyroid hormone (PTH), prostaglandin E2 (PGE2), 1-alpha-hydroxyvitamin-D3 and thrombin as compared to the response in bones which were exposed to the stimulants directly after dissection. Preincubation in basic medium did not enhance the subsequent response to PTH. By using a preincubation period in indomethacin, the dose-response curves for the stimulatory effect of PTH and PGE2 on mineral mobilization could be sensitized as compared to the curves obtained with fresh bones. Thus, the concentration of agonists causing 50% stimulation of 45Ca release was decreased by a factor of 10. The threshold for actions of PTH and PGE2 on 45Ca release was 0.01-0.03 and 1-3 nmol/l, respectively.

Transforming growth factor-beta stimulates bone resorption in neonatal mouse calvariae by a prostaglandin-unrelated but cell proliferation-dependent pathway.

The relationships between bone resorption, prostanoid formation, and cell proliferation in cultured neonatal mouse calvariae stimulated with transforming growth factor-beta (TGF-beta) have been examined. Bone resorption was assessed by analyzing the mobilization of minerals (45Ca, Ca2+., Pi) and the release of 3H from bones prelabeled with [3H]proline. Prostanoid formation was determined by analyzing the amounts of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (the stable breakdown product of PGI2) in culture media. Purified porcine TGF-beta 1 and recombinant human TGF-beta 2 stimulated the release of 45Ca and the formation of prostanoids. The effects were time and dose dependent. The concentrations of TGF-beta 1 and TGF-beta 2 causing half maximal stimulation of 45Ca release were 1 and 0.1 ng/ml, respectively. TGF-beta 1 also enhanced the release of 3H from [3H]proline labeled bones and the mobilization of Ca2+ and Pi from unlabeled bones, as well as the release of lysosomal enzymes (beta-N-acetylglucosaminidase). The degree of stimulation of mineral mobilization and matrix degradation was less than that obtained in bones stimulated with parathyroid hormone or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). TGF-beta 1-induced stimulation of 45Ca release was inhibited by calcitonin, acetazolamide, and the biphosphonate AHPrBP, three different osteoclast inhibitors. In contrast to the escape from calcitonin-induced inhibition seen in parathyroid hormone (PTH)-stimulated bones, the inhibitory effect of calcitonin in TGF-beta 1-stimulated bones persisted in long-term cultures (144 h). The stimulatory effect of TGF-beta 1 was inhibited by anti-TGF-beta 1 and by gamma-interferon (1000 U/ml). Indomethacin (1 microM), flurbiprofen (1 microM), and meclofenamic acid (1 microM) completely abolished the stimulatory effect of TGF-beta 1 on PGE2 and 6-keto-PGF 1 alpha formation without affecting TGF-beta 1-induced stimulation of 45Ca release. Similarly, the stimulatory effect of TGF-beta 2 on 45Ca release was unaffected by indomethacin. In bones in which prostaglandin formation was abolished by indomethacin, a 45Ca release response to TGF-beta 1 was obtained at 12 h. The mitotic inhibitor hydroxyurea inhibited TGF-beta 1 but not PTH-induced 45Ca release. These data demonstrate that TGF-beta 1 and TGF-beta 2 have the capacity to stimulate bone resorption and prostanoid formation in neonatal mouse calvariae, but that the effect of TGF-beta on bone resorption is unrelated to prostanoid formation. In addition, it is shown that bone resorption stimulated by TGF-beta is dependent on cell replication.

Tumor necrosis factors alpha and beta can stimulate bone resorption in cultured mouse calvariae by a prostaglandin-independent mechanism.

Human recombinant tumor necrosis factors alpha and beta (TNF-alpha and TNF-beta), at and above 1 ng/ml (approximately equal to 70 pM), caused a dose- and time-dependent enhancement of 45Ca release from neonatal mouse calvarial bones in vitro. In addition, TNF-alpha and TNF-beta (3-100 ng/ml) caused a dose-dependent stimulation of prostaglandin E2 (PGE2) formation in the calvarial bones. TNF-alpha also enhanced the biosynthesis of PGI2, as assessed by analysis of the stable breakdown product 6-keto-PGF1 alpha. The stimulatory actions of TNF-alpha and TNF-beta on PGE2 formation was maximal at 12 h. Indomethacin, flurbiprofen, and meclofenamic acid, three structurally unrelated nonsteroidal antiinflammatory drugs, abolished PGE2 biosynthesis induced by TNF-alpha and TNF-beta (100 ng/ml). The 45Ca release stimulated by TNF-alpha and TNF-beta (100 ng/ml), however, was only slightly reduced by indomethacin, flurbiprofen, and meclofenamic acid. The partial inhibitory effect of indomethacin on 45Ca release was seen over a wide range of TNF-alpha concentrations, without affecting the concentration producing half-maximal stimulatory response. TNF-alpha and TNF-beta (100 ng/ml) stimulated bone matrix breakdown, as assessed by analysis of the release of 3H from bone prelabeled with [3H]proline. Also, the stimulatory effect of TNF-alpha and TNF-beta on bone matrix degradation was partially reduced by indomethacin. Hydrocortisone (1 microM) and dexamethasone (0.1 microM) abolished TNF-alpha- and TNF-beta-induced production of PGE2. In contrast to the cyclooxygenase inhibitors, the corticosteroids did not affect the stimulatory action by the cytokines on 45Ca release.(ABSTRACT TRUNCATED AT 250 WORDS)