Fetal bone resorption was measured by an organ culture technique using fetuses from thyroparathyroidectomized (TPTX) pregnant rats to investigate the roles of fetal and maternal hormones in the regulation of basal bone resorption in utero. Thyroparathyroidectomized female rats were treated with thyroxine and/or metabolites of vitamin D3. Basal bone resorption was greatly increased in fetuses from TPTX mothers. Administration of 1,25-dihydroxycholecalciferol or 1,24,25-trihydroxycholecalciferol to TPTX female rats normalized the basal bone resorption of the fetus whereas injection of 24,25-dihydroxycholecalciferol was without effect. Treatment of the TPTX mothers with thyroxine was also shown to normalize basal bone resorption in the fetus.
JEAN-MICHEL GAREL, CHARLYNE REBUT-BONNETON, and FLORIAN DELBARRE
Muneaki Ishijima, Kunikazu Tsuji, Susan R Rittling, Teruhito Yamashita, Hisashi Kurosawa, David T Denhardt, Akira Nifuji, Yoichi Ezura, and Masaki Noda
Mechanical stress to bone plays a crucial role in the maintenance of bone homeostasis. It causes the deformation of bone matrix and generates strain force, which could initiate the mechano-transduction pathway. The presence of osteopontin (OPN), which is one of the abundant proteins in bone matrix, is required for the effects of mechanical stress on bone, as we have reported that OPN-null (OPN−/−) mice showed resistance to unloading-induced bone loss. However, cellular mechanisms underlying the phenomenon have not been completely elucidated. To obtain further insight into the role of OPN in mediating mechanical stress effect on bone, we examined in vitro mineralization and osteoclast-like cell formation in bone marrow cells obtained from hind limb bones of OPN−/− mice after tail suspension. The levels of mineralized nodule formation of bone marrow cells derived from the femora subjected to unloading were decreased compared with that from loaded control in wild-type mice. However, these were not decreased in cells from OPN−/− mice after tail suspension compared with that from loaded OPN−/− mice. Moreover, while spreading of osteoclast-like cells derived from bone marrow cells of the femora subjected to unloading was enhanced compared with that from loaded control in wild-type mice, this enhancement of spreading of these cells derived from the femora subjected to unloading was not recognized compared with those from loaded control in OPN−/− mice. These data provided cellular bases for the effect of the OPN deficiency on in vitro reduced mineralized nodule formation by osteoblasts and on enhancement of osteoclast spreading in vitro induced by the absence of mechanical stress. These in vitro results correlate well with the resistance to unloading-induced bone loss in OPN−/− mice in vivo, suggesting that OPN has an important role in the effects of unloading-induced alterations of differentiation of bone marrow into osteoblasts and osteoclasts.
S Boonen, J Aerssens, and J Dequeker
Age is the most important empirical determinant of bone mass. Factors associated with ageing account for the slow phase of bone loss, which begins in cortical bone by the age of 40 and continues throughout life at a rate of about 0·6% per year. In trabecular bone, it may begin even earlier and continues at a rate of about 0·7% per year (Burger et al. 1994, Greenspan et al. 1994, May et al. 1994). Although this loss probably reflects the aggregate effects of several processes, bone mass and bone strength depend, in part, upon calcium balance. When the amount of calcium absorbed from the diet is insufficient, calcium must be withdrawn from bone, which contains 99% of the total body stores. In both sexes, active intestinal transport of calcium decreases with ageing, particularly after the age of 70. This decrease is partially accounted for by an age-related deficiency of
Hong Liu, Jian Guo, Lin Wang, Ning Chen, Andrew Karaplis, David Goltzman, and Dengshun Miao
To assess the roles of 1,25-dihydroxyvitamin D (1,25(OH)2D) and parathyroid hormone (PTH) in hard tissue formation in oro-facial tissues, we examined the effect of either 1,25(OH)2D or PTH deficiency on dentin and dental alveolar bone formation and mineralization in the mandibles, and osteoblastic bone formation in long bones of 1α-hydroxylase knockout (1α(OH)ase−/−) mice. Compared with wild-type mice, the mineral density was decreased in the teeth and mandibles, and unmineralized dentin (predentin and biglycan immunopositive dentin) and unmineralized bone matrix in the dental alveolar bone were increased in 1α(OH)ase−/− mice. The dental volume, reparative dentin volume, and dentin sialoprotein immunopositive areas were reduced in 1α(OH)ase−/− mice. The cortical thickness, dental alveolar bone volume, and osteoblast number were all decreased significantly in the mandibles; in contrast, the osteoblast number and surface were increased in the trabecular bone of the tibiae in 1α(OH)ase−/− mice consistent with their secondary hyperparathyroidism. The expression of PTH receptor and IGF1 was reduced slightly in mandibles, but enhanced significantly in the long bones in the 1α(OH)ase−/− mice. To control for the role of secondary hyperparathyroidism, we also examined teeth and mandibles in 6-week-old PTH−/− mice. In these animals, dental and bone volumes in mandibles were not altered when compared with their wild-type littermates. These results suggest that 1,25(OH)2D3 plays an anabolic role in both dentin and dental alveolar bone as it does in long bones, whereas PTH acts predominantly in long bones rather than mandibular bone.
Donlaporn Kittivanichkul, Narattaphol Charoenphandhu, Phisit Khemawoot, and Suchinda Malaivijitnond
Since the in vitro and in vivo anti-osteoporotic effects of Pueraria mirifica (PM) in rodents have been verified, its activity in menopausal monkeys was evaluated as required before it can be applicable for human use. In this study, postmenopausal osteoporotic monkeys were divided into two groups (five per group), and fed daily with standard diet alone (PMP0 group) or diet mixed with 1000 mg/kg body weight (BW) of PM powder (PMP1000 group) for 16 months. Every 2 months, the bone mineral density (BMD), bone mineral content (BMC) and bone geometry parameters (cortical area and thickness and periosteal and endosteal circumference) at the distal radius and proximal tibia were determined using peripheral quantitative computed tomography together with plasma and urinary bone markers. Compared with the baseline (month 0) values, the cortical, but not trabecular, BMDs and BMCs and the cortical area and thickness at the metaphysis and diaphysis of the radius and tibia of the PMP0 group continuously decreased during the 16-month study period. In contrast, PMP1000 treatment ameliorated the bone loss mainly at the cortical diaphysis by decreasing bone turnover, as indicated by the lowered plasma bone-specific alkaline phosphatase and osteocalcin levels. Generally, changes in the cortical bone geometry were in the opposite direction to the cortical bone mass after PMP1000 treatment. This study indicated that postmenopausal monkeys continuously lose their cortical bone compartment, and they have a higher possibility for long bone fractures. Oral PMP treatment could improve both the bone quantity (BMC and BMD) and quality (bone geometry).
Koichiro Komatsu, Akemi Shimada, Tatsuya Shibata, Satoshi Wada, Hisashi Ideno, Kazuhisa Nakashima, Norio Amizuka, Masaki Noda, and Akira Nifuji
Bisphosphonates (BPs) are a major class of antiresorptive drug, and their molecular mechanisms of antiresorptive action have been extensively studied. Recent studies have suggested that BPs target bone-forming cells as well as bone-resorbing cells. We previously demonstrated that local application of a nitrogen-containing BP (N-BP), alendronate (ALN), for a short period of time increased bone tissue in a rat tooth replantation model. Here, we investigated cellular mechanisms of bone formation by ALN. Bone histomorphometry confirmed that bone formation was increased by local application of ALN. ALN increased proliferation of bone-forming cells residing on the bone surface, whereas it suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment. In vitro studies revealed that pulse treatment with ALN promoted osteocalcin expression. To track the target cells of N-BPs, we applied fluorescence-labeled ALN (F-ALN) in vivo and in vitro. F-ALN was taken into bone-forming cells both in vivo and in vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiation in vitro and it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formation in vivo. DC also blocked the inhibition of Rap1A prenylation by ALN in the osteoblastic cells. These data suggest that local application of ALN promotes bone formation by stimulating proliferation and differentiation of bone-forming cells as well as inhibiting osteoclast function. These effects may occur through endocytic incorporation of ALN and subsequent inhibition of protein prenylation.
J Cruickshank, D I Grossman, R K Peng, T R Famula, and A M Oberbauer
Linear bone growth depends upon proliferation, maturation, and apoptosis of growth plate chondrocytes, processes regulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I). To investigate the contribution of GH, IGF-I and apoptosis to growth plate function, the expression of GH receptor (GHR) and IGF-I receptor (IGF-IR) mRNA were evaluated by in situ hybridization in fractionated costochondral growth plates of growing rats (at 2, 4, and 7 weeks). Apoptosis was determined by TUNEL assay and morphology in histological sections. GHR mRNA was greatest in resting cells with hypertropic cells increasing GHR expression with increasing age. Hypertropic and resting cell IGF-IR mRNA declined over the ages studied. Receptor mRNA expression was altered by exposing cells to GH or IGF-I. GH and IGF significantly decreased GHR mRNA in proliferative cells. GH and IGF also decreased IGF-IR mRNA in resting cells and the 2- and 4-week-old proliferative and hypertropic cells. Treating cells in culture with GH increased the number of apoptotic cells across all ages and zones. Histologically, apoptotic cells were observed at the chondro-osseous junction and within actively proliferating chondrocytes but not in resting cells. Apoptosis was highest at 4 weeks of age with lateral regions displaying the greatest number of cells undergoing apoptosis. These data indicate that apoptosis plays a role in growth plate function, particularly spatial configuration as indicated by the preferential lateral cell apoptosis. The susceptibility of proliferative cells to GHR and IGF-IR down regulation during the period of greatest apoptosis supports a role for the GH–IGF axis in both proliferation and apoptosis during growth plate development.
R Hatton, M Stimpel, and T J Chambers
During bone resorption, osteoclasts are closely associated with endothelial cells. The latter are able to produce several agents that regulate bone resorption. In view of the increasing evidence that angiotensin II, which can be generated by endothelial cells, has actions outside the traditional renin-angiotensin system, we tested the effect of angiotensin II on bone resorption. Angiotensin II showed no effect either on osteoclast formation or on bone resorption by isolated osteoclasts. However, in co-cultures of osteoclasts with calvarial or MC3T3-E1 osteoblastic cells, and in osteoclastic cultures co-cultured with other bone cells obtained by prolonged sedimentation, angiotensin II stimulated bone resorption to a similar degree to that observed with 1,25(OH)2 vitamin D3. Stimulation of resorption was noted at concentrations of 10−7 m and above. We found that angiotensin I also stimulated bone resorption in co-cultures of osteoclasts with osteoblastic cells, and that this action was inhibited by inhibitors of angiotensin-converting enzyme. These results identify angiotensin I and II as potent stimulators of osteoclastic bone resorption, and raise the possibility that bone might contain a tissue-renin-angiotensin system that might play a role in the regulation of bone resorption.
Journal of Endocrinology (1997) 152, 5–10
Jessica L Pierce, Ke-Hong Ding, Jianrui Xu, Anuj K Sharma, Kanglun Yu, Natalia del Mazo Arbona, Zuleika Rodríguez-Santos, Paul J Bernard, Wendy B Bollag, Maribeth H Johnson, Mark W Hamrick, Dana L Begun, Xing-Ming Shi, Carlos M Isales, and Meghan E McGee-Lawrence
Excess fat within bone marrow is associated with lower bone density. Metabolic stressors such as chronic caloric restriction (CR) can exacerbate marrow adiposity, and increased glucocorticoid signaling and adrenergic signaling are implicated in this phenotype. The current study tested the role of glucocorticoid signaling in CR-induced stress by conditionally deleting the glucocorticoid receptor (Nr3c1; hereafter abbreviated as GR) in bone marrow osteoprogenitors (Osx1-Cre) of mice subjected to CR and ad libitum diets. Conditional knockout of the GR (GR-CKO) reduced cortical and trabecular bone mass as compared to WT mice under both ad libitum feeding and CR conditions. No interaction was detected between genotype and diet, suggesting that the GR is not required for CR-induced skeletal changes. The lower bone mass in GR-CKO mice, and the further decrease in bone by CR, resulted from suppressed bone formation. Interestingly, treatment with the β-adrenergic receptor antagonist propranolol mildly but selectively improved metrics of cortical bone mass in GR-CKO mice during CR, suggesting interaction between adrenergic and glucocorticoid signaling pathways that affects cortical bone. GR-CKO mice dramatically increased marrow fat under both ad libitum and CR-fed conditions, and surprisingly propranolol treatment was unable to rescue CR-induced marrow fat in either WT or GR-CKO mice. Additionally, serum corticosterone levels were selectively elevated in GR-CKO mice with CR, suggesting the possibility of bone–hypothalamus–pituitary–adrenal crosstalk during metabolic stress. This work highlights the complexities of glucocorticoid and β-adrenergic signaling in stress-induced changes in bone mass, and the importance of GR function in suppressing marrow adipogenesis while maintaining healthy bone mass.
S Keila, A Kelner, and M Weinreb
Prostaglandin E(2) (PGE(2)) has been shown to exert a bone anabolic effect in young and adult rats. In this study we tested whether it possesses a similar effect on bone formation and bone mass in aging rats. Fifteen-month-old rats were injected daily with either PGE(2) at 5 mg/kg or vehicle for 14 days. PGE(2) treatment stimulated the rate of cancellous bone formation (a approximately 5.5-fold increase in bone formation rate), measured by the incorporation of calcein into bone-forming surfaces at the tibial proximal metaphysis. This effect resulted in increased cancellous bone area (+54%) at the same site. Since PGE(2) treatment resulted in a much higher proportion of bone surface undergoing bone formation and thus lined with osteoblasts, we tested the hypothesis that PGE(2) stimulates osteoblast differentiation from bone marrow precursor cells both in vivo and in vitro. We found that ex vivo cultures of bone marrow stromal cells from rats injected for 2 weeks with PGE(2) at 5 mg/kg per day yielded more ( approximately 4-fold) mineralized nodules and exhibited a greater (by 30-40%) alkaline phosphatase activity compared with cultures from vehicle-injected rats, attesting to a stimulation of osteoblastic differentiation by PGE(2). We also compared the osteogenic capacity of bone marrow from aging (15-month-old) versus young (5-week-old) rats and its regulation by PGE(2) in vitro. Bone marrow stromal cell cultures from aging rats exhibited a greatly diminished osteogenic capacity, reflected in reduced nodule formation ( approximately 6% of young animals) and lower alkaline phosphatase activity ( approximately 60% of young animals). However, these parameters could be stimulated in both groups of animals by incubation with 10-100 nM PGE(2). The magnitude of this stimulation was greater in cultures from aging rats (+550% vs +70% in nodule formation of aging compared with young rats). In conclusion, we demonstrate here that PGE(2) exerts a bone anabolic effect in aging rats, similar to the effect we and others have reported in young, growing rats. The PGE(2)-stimulated bone formation, which augments bone mass, most likely results from recruitment of osteoblasts from their bone marrow stromal precursors.