Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.     

Thyroid-stimulating hormone maintains bone mass and strength by suppressing osteoclast differentiation

It has been suggested that pituitary hormone might be associated with bone metabolism. To investigate the role of thyroid-stimulating hormone (TSH) in bone metabolism, we designed the present study as follows. After weaning, TSH receptor (TSHR) null mice (Tshr^−/−) were randomly divided into a thyroxine treatment group (n=10) or non-treatment group (n=10); the treatment group received a dose of desiccated thyroid extract at 100 ppm daily for 5 weeks. Age-matched wild-type (Tshr^+/+, n=10) and heterozygote mice (Tshr^+/−, n=10) served as controls. After 5 weeks, the animals were sacrificed, and the femurs were collected for histomorphometrical and biomechanical analyses. In addition, the effect of TSH on osteoclastogenesis was examined in the RAW264.7 osteoclast cell line. We found that compared with Tshr^+/+ mice, Tshr^−/− and Tshr^+/− mice had lower bone strength. The histomorphometric results showed that trabecular bone volume, osteoid surface, osteoid thickness and osteoblast surface were significantly decreased, whereas the osteoclast surface was significantly increased in both Tshr^−/− and Tshr^+/− mice compared with Tshr^+/+ mice. Bone resorption and formation in Tshr^−/− mice were further enhanced by thyroxine replacement. bTSH inhibited osteoclast differentiation in vitro, as demonstrated by reduced development of TRAP-positive cells and down-regulation of differentiation markers, including tartrate-resistant acid phosphatase, matrix metallo-proteinase-9 and cathepsin K in RAW264.7 cells. Our results confirm that TSH increased bone volume and improved bone microarchitecture and strength at least partly by inhibiting osteoclastogenesis.     

Resorption of vital or devitalized bone by isolated osteoclasts in vitro

Summary The maintainance of resorptive capability towards vital or devitalized bone in osteoclasts isolated from the medullary bone of laying hens and cultured for five days in vitro has been investigated morphologically with the aid of light and transmission electron microscopy. Devitalized bone particles ranging in size from 50 to 100 m, added to cultures of osteoclasts, were rapidly surrounded by the osteoclasts which, in transmission electron microscopy, showed ruffled borders and clear zones at the surfaces of contact with bone — features typical of resorptive activity. Alternatively osteoclasts were added onto the endosteal surfaces of vital or devitalized diaphyses of quail femurs after removal of the endosteal and periosteal cell layers. The results indicated that, when the vital or devitalized bone surfaces were devoid of cells, the osteoclasts adhered and resorbed bone (as confirmed by transmission electron microscopy). When vital bone of quail was cultured for 24 h before the addition of osteoclasts a new cell layer was formed; it enveloped all bone surfaces and precluded the access of osteoclasts to bone. The role of these lining cells, ultrastructurally indistinguishable from resting osteoblasts, is discussed.     

Novel aspects of osteoclast activation and osteoblast inhibition in myeloma bone disease

Increased bone resorption is a major characteristic of multiple myeloma and is caused by osteoclast activation and osteoblast inhibition (uncoupling). Myeloma cells alter the local regulation of bone metabolism by increasing the receptor activator of NF-kappaB ligand (RANKL) and decreasing osteoprotegerin expression within the bone marrow microenvironment, thereby stimulating the central pathway for osteoclast formation and activation. In addition, they produce the chemokines MIP-1alpha, MIP-1beta, and SDF-1alpha, which also increase osteoclast activity. On the other hand, myeloma cells suppress osteoblast function by the secretion of osteoblast inhibiting factors, e.g., the Wnt inhibitors DKK-1 and sFRP-2. Moreover, they inhibit differentiation of osteoblast precursors and induce apoptosis in osteoblasts. The resulting bone destruction releases several cytokines, which in turn promote myeloma cell growth. Therefore, the inhibition of bone resorption could stop this vicious circle and not only decrease myeloma bone disease, but also the tumor progression.     

Adiponectin induces the anti-inflammatory cytokines IL-10 and IL-1RA in human leukocytes

There is increasing evidence that the adipose tissue and immunologic processes are closely linked. The most abundant protein within the adipocyte is adiponectin. Our current work reports that adiponectin has potent immuno-suppressive properties, as it induces the production of the anti-inflammatory mediators IL-10 and IL-1RA in primary human monocytes, monocyte-derived macrophages, and dendritic cells. In addition, adiponectin significantly impaired the production of the pro-inflammatory cytokine IFN-gamma in human macrophages. Moreover, adiponectin-treated macrophages exhibit a reduced phagocytotic and allo-stimulatory capacity. However, we could not detect any functional deficits or phenotypic changes in adiponectin-treated monocytes and monocyte-derived DC. In summary, the presented data support the idea that adiponectin might be of critical relevance for cytokine regulation in obesity and fatty liver diseases affecting primarily macrophage functions. This might represent a fundamental link between over-nutrition and an impaired inflammatory immune response.     

Effects of increased hypothalamic leptin gene expression on ovariectomy-induced bone loss in rats

Estrogen deficiency results in accelerated bone turnover with a net increase in bone resorption. Subcutaneous administration of leptin attenuates bone loss in ovariectomized (ovx) rats by reducing bone resorption. However, in addition to its direct beneficial effects, leptin has been reported to have indirect (central nervous system-mediated) antiosteogenic effects on bone, which may limit the efficacy of elevated serum leptin to prevent estrogen deficiency-associated bone loss. The present study evaluated the long-term effects of increased hypothalamic leptin transgene expression, using recombinant adeno-associated virus-leptin (rAAV-Lep) gene therapy, on bone mass, architecture, and cellular endpoints in sexually mature ovx Sprague-Dawley rats. Ovx rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained for 10 weeks. Additional controls consisted of ovary-intact rats and ovx rats pair-fed to rAAV-Lep rats. Lumbar vertebrae were analyzed by micro-computed tomography and tibiae by histomorphometry. Cancellous bone volume was lower and osteoclast perimeter, osteoblast perimeter, and bone marrow adipocyte density were greater in ovx rats compared to ovary-intact controls. In contrast, differences among ovx groups were not detected for any endpoint evaluated. In conclusion, whereas estrogen deficiency resulted in marked cancellous osteopenia, increased bone turnover and marrow adiposity, increasing hypothalamic leptin transgene expression in ovx rats had neither detrimental nor beneficial effects on bone mass, architecture, or cellular endpoints. These findings demonstrate that the antiresorptive effects of subcutaneous leptin administration in ovx rats are mediated through leptin targets in the periphery.     

Modeling the interactions between osteoblast and osteoclast activities in bone remodeling

We propose a mathematical model explaining the interactions between osteoblasts and osteoclasts, two cell types specialized in the maintenance of the bone integrity. Bone is a dynamic, living tissue whose structure and shape continuously evolves during life. It has the ability to change architecture by removal of old bone and replacement with newly formed bone in a localized process called remodeling. The model described here is based on the idea that the relative proportions of immature and mature osteoblasts control the degree of osteoclastic activity. In addition, osteoclasts control osteoblasts differentially depending on their stage of differentiation. Despite the tremendous complexity of the bone regulatory system and its fragmentary understanding, we obtain surprisingly good correlations between the model simulations and the experimental observations extracted from the literature. The model results corroborate all behaviors of the bone remodeling system that we have simulated, including the tight coupling between osteoblasts and osteoclasts, the catabolic effect induced by continuous administration of PTH, the catabolic action of RANKL, as well as its reversal by soluble antagonist OPG. The model is also able to simulate metabolic bone diseases such as estrogen deficiency, vitamin D deficiency, senescence and glucocorticoid excess. Conversely, possible routes for therapeutic interventions are tested and evaluated. Our model confirms that anti-resorptive therapies are unable to partially restore bone loss, whereas bone formation therapies yield better results. The model enables us to determine and evaluate potential therapies based on their efficacy. In particular, the model predicts that combinations of anti-resorptive and anabolic therapies provide significant benefits compared with monotherapy, especially for certain type of skeletal disease. Finally, the model clearly indicates that increasing the size of the pool of preosteoblasts is an essential ingredient for the therapeutic manipulation of bone formation. This model was conceived as the first step in a bone turnover modeling platform. These initial modeling results are extremely encouraging and lead us to proceed with additional explorations into bone turnover and skeletal remodeling.     

Adiponectin is regulated differently by chronic exercise than by weight-matched food restriction in hyperphagic and obese OLETF rats

This study was intended to investigate the effects of chronic exercise on blood adiponectin level. Male Otsuka Long Evans Tokushima Fatty (OLETF) rats (26 weeks old) were divided to undergo either regular 12-week wheel running exercise (EX) or to have food restriction (FR) that resulted in body weight reduction similar to that in EX. Both EX and FR induced similar reductions in body weight, abdominal fat volume and plasma leptin concentration compared with ad libitum control. At the end of the study, although plasma adiponectin level was increased in FR, the adiponectin level did not change in EX. Plasma testosterone level was higher in EX than in either of the other two groups. A significant inverse relationship existed between plasma levels of adiponectin and testosterone for all groups. Our results suggested that 12-week voluntary wheel running exercise induces different effects on plasma adiponectin level than does food restriction, despite similar reduction in body weight, fat tissue mass and plasma leptin concentration. We speculate that the elevated plasma testosterone concentration might offset any hyperadiponectinemic effect of body weight and fat volume reduction in exercising rats.     

Increased bone mass in mice after single injection of anti-receptor activator of nuclear factor-kappaB ligand-neutralizing antibody: evidence for bone anabolic effect of parathyroid hormone in mice with few osteoclasts

Receptor activator of nuclear factor-κB ligand (RANKL) is a pivotal osteoclast differentiation factor. To investigate the effect of RANKL inhibition in normal mice, we prepared an anti-mouse RANKL-neutralizing monoclonal antibody (Mab, clone OYC1) and established a new mouse model with high bone mass induced by administration of OYC1. A single subcutaneous injection of 5 mg/kg OYC1 in normal mice significantly augmented the bone mineral density in the distal femoral metaphysis from day 2 to day 28. The OYC1 treatment markedly reduced the serum level of tartrate-resistant acid phosphatase-5b (TRAP-5b, a marker for osteoclasts) on day 1, and this level was undetectable from day 3 to day 28. The serum level of alkaline phosphatase (a marker for osteoblasts) declined significantly following the reduction of TRAP-5b. Histological analysis revealed few osteoclasts in femurs of the treated mice on day 4, and both osteoclasts and osteoblasts were markedly diminished on day 14. Daily injection of parathyroid hormone for 2 weeks increased the bone mineral density in trabecular and cortical bone by stimulating bone formation in the OYC1-treated mice. These results suggest that parathyroid hormone exerted its bone anabolic activity in mice with few osteoclasts. The mouse anti-RANKL neutralizing antibody OYC1 may be a useful tool to investigate unknown functions of RANKL in vivo.     

Reduced adipose tissue mass and hypoleptinemia in iNOS deficient mice: effect of LPS on plasma leptin and adiponectin concentrations

The AAA+ chaperone ClpB solubilizes in cooperation with the DnaK chaperone system aggregated proteins. The mechanistic features of the protein disaggregation process are poorly understood. Here, we investigated the mechanism of ClpB/DnaK-dependent solubilization of heat-aggregated malate dehydrogenase (MDH) by following characteristics of MDH aggregates during the disaggregation reaction. We demonstrate that disaggregation is achieved by the continuous extraction of unfolded MDH molecules and not by fragmentation of large MDH aggregates. These findings support a ClpB-dependent threading mechanism as an integral part of the disaggregation reaction.     

Adiponectin is synthesized and secreted by human and murine cardiomyocytes

Adiponectin is thought to play a decisive role in the relationships among obesity, insulin resistance and cardiovascular risk. This study investigated whether cardiomyocytes synthesize and secrete adiponectin, and the effects of this hormone on cardiac cells. RT-PCR showed that mouse, rat and human cardiomyocytes produced mRNA for adiponectin and adiponectin receptors 1 and 2. Immunohistochemistry confirmed the presence of adiponectin in the cytoplasm of cultured cardiomyocytes, and radioimmunoassay showed that these cells secreted adiponectin into the culture medium. Exogenous adiponectin enhanced glucose and fatty acid uptake and induced AMPK phosphorylation in cultured cardiomyocytes. Our results demonstrate that adiponectin is synthesized and secreted by isolated murine and human cardiomyocytes, and suggest that the local production of this hormone by cardiomyocytes could be involved in the regulation of cardiac metabolism and function.     

Alendronate and Pamidronate calcium phosphate bone cements: setting properties and in vitro response of osteoblast and osteoclast cells

We have investigated the effect of Alendronate and Pamidronate, two bisphosphonates widely employed for the treatment of pathologies related to bone loss, on the setting properties and in vitro bioactivity of a calcium phosphate bone cement. The cement composition includes α-tricalcium phosphate (α-TCP) (90 wt%), nanocrystalline hydroxyapatite (5 wt%) and CaHPO₄ · 2H₂O (5 wt%). Disodium Alendronate and disodium Pamidronate were added to the liquid phase (bidistilled water) at two different concentrations: 0.4 and 1 mM (AL0.4, AL1.0, PAM0.4, PAM1.0). Both the initial and the final setting times of the bisphosphonate-containing cements increase with respect to the control cement. X-ray diffraction analysis, mechanical tests, and SEM investigations were carried out on the cements after different times of soaking in physiological solution. The rate of transformation of α-TCP into calcium deficient hydroxyapatite, as well as the microstructure of the cements, is not affected by the presence of Alendronate and Pamidronate. At variance, the bisphosphonates provoke a modest worsening of the mechanical properties. MG63 osteoblasts grown on the cements show a normal morphology and biological tests demonstrate very good rate of proliferation and viability in every experimental time. In particular, both Alendronate and Pamidronate promote osteoblast proliferation and differentiation, whereas they inhibit osteoclastogenesis and osteoclast function.     

Paradoxical bilateral asymmetry in bone size and bone mass in the hand

Among 227 chronic renal disease patients, micrometer caliper radiogrammetric measurements of the second metacarpal at midshaft showed the right metacarpal to be larger (with greater bone area) and with a greater cortical area than the left second metacarpal, both in 208 right-handed individuals and in 19 left-handed individuals. This direction of asymmetry was individually characteristic of the majority of individuals, whether right-handed, left-handed or ambidextrous.     

Adiponectin inhibits osteoclastogenesis and bone resorption via APPL1-mediated suppression of Akt1

Adiponectin is an adipokine playing an important role in regulating energy homeostasis and insulin sensitivity. However, the effect of adiponectin on bone metabolism shows contradictory results according to different research studies. In this study femurs were isolated from genetically double-labeled mBSP9.0Luc/β-ACT-EGFP transgenic mice and were transplanted into adiponectin knock-out mice or wild type mice to investigate the effect of temporary exposure to adiponectin deficiency on bone growth and metabolism. We found that the growth of bone explants in adiponectin knock-out mice was significantly retarded. Histological analysis, microcomputed tomography analysis, and tartrate-resistant acid phosphatase staining revealed reduced trabecular bone volume, decreased cortical bone, and increased osteoclast number in bone explants in adiponectin knock-out mice. We then found that adiponectin inhibits RANKL-induced osteoclastogenesis from RAW264.7 cells and down-regulates RANKL-enhanced expressions of osteoclastogenic regulators including NFAT2, TRAF6, cathepsin K, and tartrate-resistant acid phosphatase. Adiponectin also increases osteoclast apoptosis and decreases survival/proliferation of osteoclast precursor cells. Using siRNA specifically targeting APPL1, the first identified adaptor protein of adiponectin signaling, we found that the inhibitory effect of adiponectin on osteoclasts was induced by APPL1-mediated down-regulation of Akt1 activity. In addition, overexpression of Akt1 successfully reversed adiponectin-induced inhibition in RANKL-stimulated osteoclast differentiation. In conclusion, adiponectin is important in maintaining the balance of energy metabolism, inflammatory responses, and bone formation.     

Fibroblast growth factor 2 stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway

Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/β-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and β-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and β-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3β, a negative regulator of Wnt/β-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted β-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.     

Retinoids stimulate periosteal bone resorption by enhancing the protein RANKL, a response inhibited by monomeric glucocorticoid receptor

Increased vitamin A (retinol) intake has been suggested to increase bone fragility. In the present study, we investigated effects of retinoids on bone resorption in cultured neonatal mouse calvarial bones and their interaction with glucocorticoids (GC). All-trans-retinoic acid (ATRA), retinol, retinalaldehyde, and 9-cis-retinoic acid stimulated release of (45)Ca from calvarial bones. The resorptive effect of ATRA was characterized by mRNA expression of genes associated with osteoclast differentiation, enhanced osteoclast number, and bone matrix degradation. In addition, the RANKL/OPG ratio was increased by ATRA, release of (45)Ca stimulated by ATRA was blocked by exogenous OPG, and mRNA expression of genes associated with bone formation was decreased by ATRA. All retinoid acid receptors (RARα/β/γ) were expressed in calvarial bones. Agonists with affinity to all receptor subtypes or specifically to RARα enhanced the release of (45)Ca and mRNA expression of Rankl, whereas agonists with affinity to RARβ/γ or RARγ had no effects. Stimulation of Rankl mRNA by ATRA was competitively inhibited by the RARα antagonist GR110. Exposure of calvarial bones to GC inhibited the stimulatory effects of ATRA on (45)Ca release and Rankl mRNA and protein expression. This inhibitory effect was reversed by the glucocorticoid receptor (GR) antagonist RU 486. Increased Rankl mRNA stimulated by ATRA was also blocked by GC in calvarial bones from mice with a GR mutation that blocks dimerization (GR(dim) mice). The data suggest that ATRA enhances periosteal bone resorption by increasing the RANKL/OPG ratio via RARα receptors, a response that can be inhibited by monomeric GR.     

Recql4 haploinsufficiency in mice leads to defects in osteoblast progenitors: Implications for low bone mass phenotype

The cellular and molecular mechanisms that underlie skeletal abnormalities in defective Recql4-related syndromes are poorly understood. Our objective in this study was to explore the function of Recql4 in osteoblast biology both in vitro and in vivo. Immunohistochemistry on adult mouse bone showed Recql4 protein localization in active osteoblasts around growth plate, but not in fully differentiated osteocytes. Consistent with this finding, Recql4 gene expression was high in proliferating mouse osteoblastic MC3T3.E1 cells and decreased as cells progressively lost their proliferation activity during differentiation. Recql4 overexpression in osteoblastic cells exhibited higher proliferation activity, while its depletion impeded cell growth. In addition, bone marrow stromal cells from male Recql4+/- mice had fewer progenitor cells, including osteoprogenitors, indicated by reduced total fibroblast colony forming units (CFU-f) and alkaline phosphatase-positive CFU-f colonies concomitant with reduced bone mass. These findings provide evidence that Recql4 functions as a regulatory protein during osteoprogenitor proliferation, a critical cellular event during skeleton development.