Reduction In Trabecular Bone Mass Research Articles

Cortactin controls bone homeostasis through regulating the differentiation of osteoblasts and osteoclasts.

Cortactin (CTTN), a cytoskeletal protein and substrate of Src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that CTTN was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that CTTN promoted the in vitro differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, CTTN was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that CTTN could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by CTTN siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by CTTN overexpression. Notably, transplantation of CTTN-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, CTTN-silenced BMSCs expressed higher levels of receptor activator of nuclear factor κB ligand (RANKL) than control BMSCs did and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that CTTN favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of CTTN expression may be advantageous for maintaining bone homeostasis.

Mitofusin 2 plays a critical role in maintaining the functional integrity of the neuromuscular-skeletal axis

PurposeMitofusin 2 (Mfn2) is one of two mitofusins involved in regulating mitochondrial size, shape and function, including mitophagy, an important cellular mechanism to limit oxidative stress. Reduced expression of Mfn2 has been associated with impaired osteoblast differentiation and function and a reduction in the number of viable osteocytes in bone. We hypothesized that the genetic absence of Mfn2 in these cells would increase their susceptibility to aging-associated metabolic stress, leading to a progressive impairment in skeletal homeostasis over time. MethodsMfn2 was selectively deleted in vivo at three different stages of osteoblast lineage commitment by crossing mice in which the Mfn2 gene was floxed with transgenic mice expressing Cre under the control of the promoter for Osterix (OSX), collagen1a1, or DMP1 (Dentin Matrix Acidic Phosphoprotein 1). ResultsMice in which Mfn2 was deleted using DMP1-cre demonstrated a progressive and dramatic decline in bone mineral density (BMD) beginning at 10 weeks of age (n = 5 for each sex and each genotype from age 10 to 20 weeks). By 15 weeks, there was evidence for a functional decline in muscle performance as assessed using a rotarod apparatus (n = 3; 2 males/ 1 female for each genotype), accompanied by a decline in lean body mass. A marked reduction in trabecular bone mass was evident on bone histomorphometry, and biomechanical testing at 25 weeks (k/o: 2 male/1 female, control 2 male/2 female) revealed severely impaired femur strength. Extensive regional myofiber atrophy and degeneration was observed on skeletal muscle histology. Electron microscopy showed progressive disruption of cellular architecture, with disorganized sarcomeres and a bloated mitochondrial reticulum. There was also evidence of neurodegeneration within the ventral horn and roots of the lumbar spinal cord, which was accompanied by myelin loss and myofiber atrophy. Deletion of Mfn2 using OSX-cre or Col1a1-cre did not result in a musculoskeletal phenotype. Where possible, male and female animals were analyzed separately, but small numbers of animals in each group limited statistical power. For other outcomes, where sex was not considered, small sample sizes might still limit the strength of the observation. ConclusionDespite known functional overlap of Mfn1 and Mfn2 in some tissues, and their co-expression in bone, muscle and spinal cord, deletion of Mfn2 using the 8 kB DMP1 promoter uncovered an important non-redundant role for Mfn2 in maintaining the neuromuscular/bone axis.

Rodent model of disuse-induced bone loss by hind limb injection with botulinum toxin A

Bone loss materializes rapidly after immobilization or mechanical unloading. Hind limb injection with botulinum toxin A (BTX) is a highly reproducible animal model for disuse-induced bone loss. Here we describe an easy-to-use and enhanced version of the method employing multiple hind limb injections with BTX to induce a pervasive muscle paralysis and thereby disuse of the hind limb. Thirty-six 12–14-week-old female Wistar rats were stratified into three groups: Baseline (Base), Control (Ctrl), and BTX. Disuse was achieved by injecting BTX directly into the right quadriceps femoris muscle, the hamstring muscles, and the posterior calf muscles. The rats were sacrificed after six weeks, and the right rectus femoris muscle and femur were isolated and analyzed. Hind limb disuse resulted in a significant and substantial loss of both muscle mass and bone mass. The loss of bone mass was accompanied by a reduction of trabecular bone mass and a deterioration of the trabecular micro-architecture with a reduction of trabecular thickness and trabecular number compared to Ctrl. In addition, the trabeculae changed from a more plate-like towards a more rod-like shape as indicated by an increase in the structure model index.•Multiple injections with BTX targeting muscles on both the anterior and posterior thigh and the calf ensure a uniform and pervasive muscle paralysis and hind limb disuse.•Hind limb injections with BTX results in a substantial loss of muscle and bone mass and deterioration of the trabecular micro-architecture.•The induction of hind limb disuse with BTX is highly reproducible.

Vdr expression in osteoclast precursors is not critical in bone homeostasis

The long-recognized role of the vitamin D endocrine system is to maintain stable serum calcium concentrations, which are ensured by a complex interplay between parathyroid gland, kidney, intestine, and bone. However, although VDR is expressed in osteoclastogenic cells, the contribution of VDR-mediated signaling to osteoclast differentiation and activity remains undefined. We therefore deleted Vdr expression efficiently and specifically in myeloid cells by use of M lysozyme-driven Cre expression, which targets granulocytes, monocytes, macrophages and osteoclasts (Vdrmyel− mice). Bone and calcium homeostasis were investigated under basal conditions and in conditions of increased bone remodeling, by feeding Vdrmyel− and Vdrmyel+ (wildtype) mice either a normal (1%) or a low (0.02%) calcium diet from weaning onwards. Vdrmyel− mice developed normally and were normocalcemic at the age of 8 weeks, both at the normal and the low calcium diet. No differences in trabecular or cortical bone mass were observed between Vdrmyel− mice and their wildtype littermates. Dietary calcium restriction resulted in a comparable reduction of trabecular bone mass (40%) and cortical thickness (48%) in Vdrmyel− and Vdrmyel+ mice, pointing to a massive transfer of calcium from the bone to the serum. In agreement with these results, osteoclastic differentiation of hematopoietic cells of Vdrmyel− mice, either induced by M-CSF and RANKL, or cocultured with osteoblasts, occurred as efficiently as osteoclastogenesis from Vdrmyel+ mice. In conclusion, our data do not support a role for osteoclastic Vdr signaling in the control of bone homeostasis.

Tsc1 Regulates the Balance Between Osteoblast and Adipocyte Differentiation Through Autophagy/Notch1/β-Catenin Cascade.

A reduction in trabecular bone mass is often associated with an increase in marrow fat in osteoporotic bones. The molecular mechanisms underlying this inverse correlation are incompletely understood. Here, we report that mice lacking tuberous sclerosis 1 (Tsc1) in Osterix-expressing cells had a significant decrease in trabecular bone mass characterized by decreased osteoblastogenesis, increased osteoclastogenesis, and increased bone marrow adiposity in vivo. In vitro study showed that Tsc1-deficient bone marrow stromal cells (BMSCs) had decreased proliferation, decreased osteogenic differentiation, and increased adipogenic differentiation in association with the downregulation of Wnt/β-catenin signaling. Mechanistically, TSC1 deficiency led to autophagy suppression and consequent Notch1 protein increase, which mediated the GSK3β-independent β-catenin degradation. Together, our results indicate that Tsc1 controls the balance between osteoblast and adipocyte differentiation of BMSCs. © 2018 American Society for Bone and Mineral Research.

Experimental repetitive mild traumatic brain injury induces deficits in trabecular bone microarchitecture and strength in mice

To evaluate the long-term consequence of repetitive mild traumatic brain injury (mTBI) on bone, mTBI was induced in 10-week-old female C57BL/6J mice using a weight drop model, once per day for 4 consecutive days at different drop heights (0.5, 1 and 1.5 m) and the skeletal phenotype was evaluated at different time points after the impact. In vivo micro-CT (μ-CT) analysis of the tibial metaphysis at 2, 8 and 12 weeks after the impact revealed a 5%–32% reduction in trabecular bone mass. Histomorphometric analyses showed a reduced bone formation rate in the secondary spongiosa of 1.5 m impacted mice at 12 weeks post impact. Apparent modulus (bone strength), was reduced by 30% (P<0.05) at the proximal tibial metaphysis in the 1.5 m drop height group at 2 and 8 weeks post impact. Ex vivo μ-CT analysis of the fifth lumbar vertebra revealed a significant reduction in trabecular bone mass at 12 weeks of age in all three drop height groups. Serum levels of osteocalcin were decreased by 22%, 15%, and 19% in the 0.5, 1.0 and 1.5 m drop height groups, respectively, at 2 weeks post impact. Serum IGF-I levels were reduced by 18%–32% in mTBI mice compared to contro1 mice at 2 weeks post impact. Serum osteocalcin and IGF-I levels correlated with trabecular BV/TV (r2=0.14 and 0.16, P<0.05). In conclusion, repetitive mTBI exerts significant negative effects on the trabecular bone microarchitecture and bone mechanical properties by influencing osteoblast function via reduced endocrine IGF-I actions.

Allogeneic yet major histocompatibility complex-matched bone marrow transplantation in mice results in an impairment of osteoblasts and a significantly reduced trabecular bone

Secondary osteopenia following allogeneic bone marrow or stem cell transplantation (BMT or HSCT) is a significant source of morbidity in patients. It is believed to be caused by a number of factors related to the myeloablative conditioning and subsequent therapy regimen. We here aimed to investigate whether the allogeneic bone marrow by itself directly impacts on the bone mass of the patient. We thus performed syn- and allogeneic BMT between two inbred mouse strains, which share an identical major histocompatibility complex background yet differ in their bone phenotypes. BMT was well tolerated, yielded survival rates of 97% and allowed for a regular physiological development. However, allogeneic BMT led to a significant reduction of trabecular bone mass that was independent of strain, sex, immunosuppressive medication, complications resulting from graft versus host disease, underlying bone phenotype and numbers of osteoclasts. Instead, reduced trabecular bone mass correlated with reduced plasma levels of amino-terminal propeptide of type I collagen. Our results suggest that osteopenia following allogeneic BMT is significantly influenced by an impaired osteoblast activity that may stem from a lack of communication between the resident osteoblasts and an allogeneic bone marrow-derived cell type. Elucidating this incompatibility will open new approaches for the therapy of secondary osteopenia.

Decreased trabecular bone deterioration of proximal tibiae and lumbar vertebrae in postmenopausal women with osteoarthritis.

Osteoporosis and osteoarthritis (OA) are two common age-related skeletal disorders, which are associated with substantial morbidity and disability, particularly among elderly women. The present study was performed to investigate the trabecular bone deterioration of proximal tibiae and lumbar vertebrae in postmenopausal women with OA. The results demonstrated that the histomorphometric section of trabecular bone below the growth plate was markedly different between the healthy control and OA group. However, the loss of trabecular bone underneath the growth plate in the healthy control group was significantly worse than that of the OA group. Hematoxylin and eosin staining demonstrated the increased disconnection and separation of the trabecular bone network as well as the reduction of trabecular bone mass of primary and secondary spongiosa throughout the proximal metaphysis of tibia in the healthy control compared to the OA group; similar results were found when the same experiment was repeated on the lumbar vertebrae of healthy control subjects and OA patients from postmenopausal women. The biological properties of trabecular bone in the proximal tibia and lumbar vertebrae were measured in postmenopausal women with OA. Spearman's rank correlation analysis revealed that the bone volume fraction was both positively correlated with radiographic severity and Western Ontario McMaster University Osteoarthritis Index scores in in the proximal tibia and lumbar vertebrae from postmenopausal women with OA. In conclusion, the structural properties of the proximal tibia and lumbar vertebrae supported that an inverse correlation existed between postmenopausal women with OA and healthy controls. Moreover, there is an important protective mechanism of OA on trabecular microstructure in proximal tibiae and lumbar vertebrae from postmenopausal women.

Bradykinin receptors and EphB2/EphrinB2 pathway in response to high glucose-induced osteoblast dysfunction and hyperglycemia-induced bone deterioration in mice.

This study was carried out in order to investigate bone dysfunction and the involvement of bradykinin receptors and the Eph/Ephrin signaling pathway in osteoblasts and in mice with diabetes-related osteoporosis in response to exposure to high glucose. Osteogenic transdifferentiation was inhibited when the osteoblasts were exposed to high glucose, and the expression levels of bone formation-related genes [Runx2 and alkaline phosphatase (ALP)] were decreased, while those of bone resorption-related genes [matrix metalloproteinase (MMP)9 and carbonic anhydrase II (CAII)] were increased. Moreover, the mRNA and protein expression levels of bradykinin receptor B1 (BK1R)/bradykinin receptor B2 (BK2R) and EphB2/EphrinB2 were significantly decreased in the osteoblasts following exposure to high glucose. Intriguingly, the interaction between BK2R and EphB2/EphrinB2 was confirmed, and BK2R loss-of-function significantly decreased the mRNA and protein expression levels of EphB2/EphrinB4. In vivo, hyperglycemia induced the disequilibrium of calcium homeostasis through the inhibition of bone formation and the acceleration of bone resorption, which was manifested by the reduction of trabecular bone mass of the primary and secondary spongiosa, as well as by the increase in the number of mature osteoclasts throughout the proximal tibial metaphysis in mice with diabetes-related osteoporosis. Furthermore, the mRNA and protein expression levels of BK1R/BK2R and EphB2/EphrinB2 in the tibias of the mice with diabetes-related osteoporosis were significantly decreased. These results demonstrate that bradykinin receptors and the EphB4/EphrinB2 pathway mediate the development of complications in mice with diabetes-related osteoporosis and suggest that the inactivation of bradykinin receptors and the EphB4/EphrinB2 pathway enhance the severity of complications in mice with diabetes-related osteoporosis.

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

The Primary Function of gp130 Signaling in Osteoblasts Is To Maintain Bone Formation and Strength, Rather Than Promote Osteoclast Formation

Interleukin-6 (IL-6) family cytokines act via gp130 in the osteoblast lineage to stimulate the formation of osteoclasts (bone resorbing cells) and the activity of osteoblasts (bone forming cells), and to inhibit expression of the osteocyte protein, sclerostin. We report here that a profound reduction in trabecular bone mass occurs both when gp130 is deleted in the entire osteoblast lineage (Osx1Cre gp130 f/f) and when this deletion is restricted to osteocytes (DMP1Cre gp130 f/f). This was caused not by an alteration in osteoclastogenesis, but by a low level of bone formation specific to the trabecular compartment. In contrast, cortical diameter increased to maintain ultimate bone strength, despite a reduction in collagen type 1 production. We conclude that osteocytic gp130 signaling is required for normal trabecular bone mass and proper cortical bone composition.

Editorial: Molecular Endocrinology Articles in the Spotlight for January 2012

Beginning with this issue of Molecular Endocrinology, we’ll be highlighting three articles that should be of special interest to our readers. Although all manuscripts accepted for publication in Molecular Endocrinology have a high degree of scientific merit, the three selected for an editorial highlight are meant to showcase particularly novel and significant findings reflective of others that can be found throughout the issue. This first issue of 2012 includes the following articles that are excellent examples of the relevance of basic science findings to clinical management of endocrine disorders. The first, “Reduced bone turnover in mice lacking the P2Y13 receptor of ADP” by Alison Gartland and colleagues, addresses the issue of osteoporosis and the need for drugs that provide alternative treatment for osteoporosis. The authors show that deletion of the P2Y13 receptor, a G protein-coupled receptor for extracellular ADP, leads to a reduction in trabecular bone mass, reduction in osteoblast and osteoclast numbers and an overall reduction in the rate of bone remodeling in mice. They also find that P2Y13R( / ) mice are protected from ovariectomyinduced bone loss. Therefore, the P2Y13 receptor may provide a new therapeutic target that confers a protective advantage in conditions of accelerated bone turnover such as estrogen deficiency-induced osteoporosis. This would be a tremendous benefit to the one third of all women and one fifth of all men over fifty who will experience an osteoporotic fracture in their lifetime. “FXR protects lung from LPS-induced acute injury”, by Wendong Huang and colleagues, is another article in the spotlight this month. This research addresses acute lung injury, which is associated with 17,000–43,000 deaths each year. In one of the important findings of this study, Huang et al. demonstrate that expression of a constitutively active FXR repressed the expression of proinflammatory genes and improved lung permeability and lung regeneration in FXR / mice. Therefore, FXR may be an ideal target for drug development as novel and safe FXR ligands may be effective for the treatments of lung injury and other related diseases. Finally, Signe Altmae and colleagues bring us a Research Resource contribution, “Interactome of human embryo implantation: identification of gene expression pathways, regulation, and integrated regulatory networks.” This study helps us to better understand the complex genetic network that leads to successful embryo implantation and will be relevant to the development of future strategies to alleviate human infertility. The authors are the first to perform thorough computational analysis to identify the potential molecular interactions between implantation-stage endometrium and the embryo. I hope you enjoy these articles, as well as the rest of the exceptional articles in this issue. With best wishes for 2012— Donald B. DeFranco, Editor-in-Chief

Reduced Bone Turnover in Mice Lacking the P2Y13 Receptor of ADP

Osteoporosis is a condition of excessive and uncoupled bone turnover, in which osteoclastic resorption exceeds osteoblastic bone formation, resulting in an overall net bone loss, bone fragility, and morbidity. Although numerous treatments have been developed to inhibit bone loss by blocking osteoclastic bone resorption, understanding of the mechanisms behind bone loss is incomplete. The purinergic signaling system is emerging to be a pivotal regulator of bone homeostasis, and extracellular ADP has previously been shown to be a powerful osteolytic agent in vitro. We report here that deletion of the P2Y(13) receptor, a G protein-coupled receptor for extracellular ADP, leads to a 40% reduction in trabecular bone mass, 50% reduction in osteoblast and osteoclast numbers in vivo, as well as activity in vitro, and an overall 50% reduction in the rate of bone remodeling in mice in vivo. Down-regulation of RhoA/ROCK I signaling and a reduced ratio of receptor activator of nuclear factor κB ligand/osteoprotegerin observed in osteoblasts from P2Y(13)R(-/-) mice might explain this bone phenotype. Furthermore, because one of the main causes of osteoporosis in older women is lack of estrogen, we examined the effect of ovariectomy of the P2Y(13)R(-/-) mice and found them to be protected from ovariectomy-induced bone loss by up to 65%. These data confirm a role of purinergic ADP signaling in the skeleton, whereby deletion of the P2Y(13) receptor leads to reduced bone turnover rates, which provide a protective advantage in conditions of accelerated bone turnover such as oestrogen deficiency-induced osteoporosis.

Selective cyclooxygenase-2 inhibitor prevents reduction of trabecular bone mass in collagen-induced arthritic mice in association with suppression of RANKL/OPG ratio and IL-6 mRNA expression in synovial tissues but not in bone marrow cells

We performed this study to clarify whether celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, prevents trabecular bone mass reduction by suppressing arthritis-related increase of bone resorption, and to discriminate differences in actions on bone among celecoxib, SC-58560 (a selective COX-1 inhibitor), and indomethacin. Eight-week-old DBA/1J male mice were divided into six groups as follows. Control untreated (Normal) and collagen-induced arthritic (CIA) mice were compared with four treatment groups: celecoxib was orally administered to CIA mice at doses of 0 (Vehicle), 16 (COX2L), and 75 (COX2H) mg/kg, in addition to two groups of mice treated with SC-58560 (COX1) or indomethacin (IND). Histomorphometry showed a significant decrease in tibial trabecular bone volume in arthritic mice, which was corrected by COX2H. The increased osteoclast surface and number in the Vehicle group were suppressed by COX2L, COX2H, and IND. The decreased bone formation rate in Vehicle was elevated by COX2H without statistical significance. A high ratio of mRNA expression of receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG) in Vehicle synovial tissue was suppressed by COX2L and COX2H. The increased expression of interleukin (IL)-6 mRNA in Vehicle was suppressed by COX2L, COX2H, and IND, although no difference in this expression was observed in bone marrow cells among all groups. In conclusion, in CIA mice, celecoxib suppresses arthritis-related increase in bone resorption at low and high doses and prevents trabecular bone mass reduction at high doses in association with suppression of osteoclast development in bone marrow through inhibition of RANKL/OPG ratio and IL-6 mRNA expression in inflammatory synovial tissue.

Osteoblast-Targeted Expression of Sfrp4 in Mice Results in Low Bone Mass

Transgenic mice overexpressing Sfrp4 in osteoblasts were established. These mice exhibited low bone mass caused by a decrease in bone formation. We recently reported that single nucleotide polymorphisms in the secreted frizzled-related protein 4 (Sfrp4) gene are responsible for low peak BMD in senescence-accelerated mouse (SAM) P6. In vitro studies revealed inhibition of osteoblast proliferation by Sfrp4, which is supposed to be mediated by canonical Wnt signaling. We examined the expression of Sfrp4 in neonate long bones by in situ hybridization and generated transgenic mice in which Sfrp4 was specifically overexpressed in osteoblasts under the control of a 2.3-kb Col1a1 osteoblast-specific promoter. Next, we compared the phenotype of Sfrp4 transgenic (Sfrp4 TG) mice with that of mice in which one allele of beta-catenin was conditionally disrupted in osteoblasts (betaChet), and administered lithium chloride (LiCl) to Sfrp4 TG mice. Hemizygous Sfrp4 TG mice exhibited a 30% reduction of trabecular bone mass compared with that in wildtype littermates at 8 wk of age, and histomorphometrical analysis showed decreases in both osteoblast numbers and bone formation rate. betaChet mice exhibited a 17% reduction of trabecular bone mass in distal femora caused by an increase in the osteoclast number and a decrease in bone formation rate. Furthermore, LiCl administration rescued the bone phenotype of Sfrp4 TG mice. Expression of Sfrp4 in periosteum and bone tissues suggested the role of Sfrp4 in osteoblasts, and we identified that overexpression of Sfrp4 in osteoblasts suppressed osteoblast proliferation, resulting in a decrease in bone formation in vivo. Partial suppression of beta-catenin/canonical Wnt signaling also impaired bone formation, and activation of the signaling restored low bone mass of Sfrp4 TG mice. Thus, these results indicate that Sfrp4 decreases bone formation at least in part by attenuating canonical Wnt signaling in vivo.

Peptide YY Regulates Bone Turnover in Rodents

Peptide YY (PYY) and pancreatic polypeptide (PPY) are members of the neuropeptide Y peptide family. The neuropeptide Y receptor signaling pathway has been implicated in a number of physiologic processes, including the regulation of energy balance and bone mass. To investigate the contribution of endogenous PYY and PPY to these processes, we generated both Pyy- and Ppy-deficient mice. Pyy(-/-) and Ppy(-/-) mice and their respective wild-type littermates were studied from 8 weeks to 9 months of age. Food intake, metabolic parameters, and locomotor activity were monitored using indirect calorimetry. Body composition and bone parameters were analyzed using dual energy x-ray absorptiometry, histomorphometry, and vertebral compression testing. Studies in these mice showed an osteopenic phenotype specific to the Pyy-deficient line, which included a reduction in trabecular bone mass and a functional deficit in bone strength. Furthermore, female Pyy(-/-) mice showed a greater sensitivity to ovariectomy-induced bone loss compared with wild-type littermates. No food intake or metabolic phenotype was apparent in male or female Pyy(-/-) mice on standard chow. However, female Pyy(-/-) mice on a high-fat diet showed a greater propensity to gain body weight and adiposity. No metabolic or osteopenic phenotype was observed in Ppy-deficient mice. These results indicate that endogenous PYY plays a critical role in regulating bone mass. In comparison, its role in regulating body weight is minor and is confined to situations of high-fat feeding.

The effect of a low-protein diet in pregnancy on offspring renal calcium handling

Low birth weight humans and rats exposed to a low-protein diet in utero have reduced bone mineral content. Renal calcium loss during the period of rapid skeletal growth is associated with bone loss. Because young rats exposed to low protein display altered renal function, we tested the hypothesis that renal calcium excretion is perturbed in this model. Pregnant Wistar rats were fed isocalorific diets containing either 18% (control) or 9% (low) protein throughout gestation. Using standard renal clearance techniques, Western blotting for renal calcium transport proteins, and assays for Na(+)-K(+)-ATPase activity and serum calcitropic hormones, we characterized calcium handling in 4-wk-old male offspring. Histomorphometric analyses of femurs revealed a reduction in trabecular bone mass in low-protein rats. Renal calcium (control vs. low protein: 10.4 +/- 2.1 vs. 27.6 +/- 4.5 nmol x min(-1) x 100 g body wt(-1); P < 0.01) and sodium excretion were increased, but glomerular filtration rate was reduced in low-protein animals. Total plasma calcium was reduced in low-protein rats (P < 0.01), but ionized calcium, serum calcitropic hormone concentrations, and total body calcium did not differ. There was no significant change in plasma membrane Ca(2+)-ATPase pump, epithelial calcium channel, or calbindin-D(28K) expression in low-protein rat kidneys. However, Na(+)-K(+)-ATPase activity was 36% lower (P < 0.05) in low-protein rats. These data suggest that the hypercalciuria of low-protein rats arises through a reduction in passive calcium reabsorption in the proximal tubule rather than active distal tubule uptake. This may contribute to the reduction in bone mass observed in this model.

Leptin Inhibits Bone Formation Not Only in Rodents, but Also in Sheep

This study examines the effect of long-term ICV administration of leptin in ewes. We found that central application significantly decreased osteoblast activity as measured by serum analysis as well as by histomorphometry, resulting in decreased trabecular bone volume. These data provide additional evidence that bone formation and therefore bone remodeling is at least in part centrally controlled. Genetic studies in mice have identified leptin as a potent inhibitor of bone formation acting through the central nervous system and unraveled the central nature of bone mass control and its disorders. Although these studies have radically enhanced our understanding of skeletal physiology because they have established a hypothalamic regulation of bone remodeling through the sympathetic nervous system, controversy remains about the physiological relevance of these observations because leptin's effect on bone after intracerebroventricular (ICV) application has only been shown in mice. To address whether leptin has a role in regulating bone mass beyond rodents, we treated ewes with long-term ICV application of leptin and analyzed the bone phenotype after a treatment period of 3 months. Three groups of corriedale sheep were compared: (1) control entire (control), (2) ovariectomy (OVX) and ICV application of cerebrospinal fluid (CSF); and (iii) OVX and ICV application of leptin (leptin). Analysis included histomorphometric characterization of iliac crest, spine and femur by histology and biomechanical testing and measurement of bone turnover parameters in serum and urine. Central application of leptin decreased bone formation by 70% and mineralizing surface (MS/BS, 39.4 +/- 3.3% versus 16.1 +/- 2.1%) significantly (p < 0.01). Whereas OVX increased osteoclast indices and urinary cross-lap excretion by two and three times, respectively, serum parameters of osteoblast activity were significantly reduced by ICV application of leptin (p < 0.01). Consequently, ewes treated with leptin were osteopenic (iliac crest BV/TV entire, 22.7 +/- 1.3%; CSF, 18.9 +/- 2.4%; leptin, 12.4 +/- 2.6%), whereas bone torsional failure load reflecting the cortex of the tibia was not yet changed after 3 months of treatment (p < 0.01). Taken together, these data suggest that leptin controls bone formation after ICV application, leading to reduction of trabecular bone mass in sheep. Most importantly, however, they show that the central regulation of bone formation is not limited to rodents, but is also found in large animals, providing further evidence that bone remodeling in vertebrates is centrally controlled.

Bone-Protecting Effect of Safflower Seeds in Ovariectomized Rats

Safflower (Carthamus tinctorius L.) seeds have long been clinically used in Korea to promote bone formation and prevent osteoporosis. However, the beneficial effect has not been scientifically evaluated. Thus, in the present study we investigated whether phytoestrogen rich safflower seeds reduce bone loss in ovariectomized rats. Female Sprague-Dawley rats were subjected to bilateral ovariectomy or sham surgery. One week after the operation, ovariectomized rats were either fed a diet containing defatted safflower seeds or injected with 17b-estradiol (E2) for 4 weeks. As expected, ovariectomy resulted in a dramatic reduction in trabecular bone mass of the proximal tibia, increase in deposition of marrow fat, and in uterine atrophy. E2 treatment almost completely prevented bone loss as well as marrow adiposity, as examined by scanning electron microscopy and histomorphometry. Safflower seeds partially prevented ovariectomy-induced bone loss and slightly reduced marrow adiposity. Safflower seeds, in contrast to E2, exerted very weak uterotrophic action. In an attempt to elucidate the underlying mechanisms, effect of polyphenolic compounds extracted from safflower seeds on proliferation of osteoblast-like cells was also assessed in vitro. The mixed polyphenolic compounds stimulated growth of ROS 17/2.8 osteoblast-like cells in a dose-dependent manner (5-100 mg/ml), as potently as E2 and genistein. The present data provide the first direct in vivo evidence that safflower seeds have a protecting effect on bone loss caused by estrogen deficiency, without substantial effect on the uterus. The beneficial effect of safflower seeds may be mediated, at least in part, by the stimulating effect of polyphenolic compounds on proliferation of osteoblasts.

Stage-dependent changes in trabecular bone turnover and osteogenic capacity of marrow cells during development of type II collagen-induced arthritis in mice

Rheumatoid arthritis (RA) is a disease characterized by inflammatory polyarthritis leading to destruction of the joints and reduction in bone mass. However, the relationship between bone mass and turnover is not yet clear in RA patients. To clarify the effect of bone turnover and marrow osteogenic capacity on mass and structure during the development of arthritis, we examined DBA1/J mice for 8 weeks after the first immunization with bovine type II collagen at the age of 9 weeks. Localized arthritis developed at 4 weeks and advanced arthritis at 6 weeks postimmunization. Urinary deoxypyridinoline levels in arthritic mice were significantly higher at 4 weeks, and levels were maintained thereafter. Their serum osteocalcin levels were significantly reduced compared with controls at 2 and 6 weeks, but did not differ significantly from those in the control group at 4 and 8 weeks. Three-dimensional (3D) trabecular bone volume of the proximal tibia measured by 3D microcomputed tomography (micro-CT) in the arthritic mice became significantly lower at 4 weeks and decreased further at 6 weeks compared with controls. Parameters of 3D trabecular bone structure, such as structure model index and trabecular bone pattern factor, were increased at 4 and 6 weeks, respectively. Trabecular osteoclast number increased and bone formation rates decreased at 8 weeks. The number of total bone marrow cells (BMCs), adherent stromal cells, and area of mineralized nodule formation in the tibia of arthritic mice were significantly reduced compared with controls at 6 weeks. Numbers of total fibroblastic colony-forming units (CFU-f) and alkaline phosphatase (ALP)-positive CFU-f colonies also decreased. However, the values of these osteogenic parameters corrected for the total BMCs and/or adherent stromal cells did not differ significantly between the arthritic and control groups. These data indicate that an increase in bone resorption led to the reduction in trabecular bone mass and deterioration of 3D structure during the localized arthritic stage. The reduction in bone marrow osteogenic potential in the advanced arthritic stage was due to the reduction in the number of total bone marrow cells, and differentiation of osteogenic cells was apparently unaffected. The reduction in bone formation may not be substantial in this arthritic model.