Role of PTH1R internalization in osteoblasts and bone mass using a phosphorylation-deficient knock-in mouse model

Phosphorylation, internalization, and desensitization of G protein-coupled receptors, such as the parathyroid hormone (PTH) and PTH-related peptide (PTHrP) receptor (PTH1R), are well characterized and known to regulate the cellular responsiveness in vitro. However, the role of PTH1R receptor phosphorylation in bone formation and osteoblast functions has not yet been elucidated. In previous studies, we demonstrated impaired internalization and sustained cAMP stimulation of a phosphorylation-deficient (pd) PTH1R in vitro, and exaggerated cAMP and calcemic responses to s.c. PTH infusion in pdPTH1R knock-in mouse model. In this study, we examined the impact of impaired PTH1R phosphorylation on the skeletal phenotype of mice maintained on normal, low, and high calcium diets. The low calcium diet moderately reduced (P<0.05) bone volume and trabecular number, and increased trabecular spacing in both wild-type (WT) and pd mice. The effects, however, seem to be less pronounced in the female pd compared to WT mice. In primary calvarial osteoblasts isolated from 2-week-old pd or WT mice, PTH and PTHrP decreased phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2), a member of mitogen-activated protein kinase, and cyclin D1, a G?/S phase cyclin, in vitro. In contrast to WT osteoblasts, down-regulation of cyclin D1 was sustained for longer periods of time in osteoblasts isolated from the pd mice. Our results suggest that adaptive responses of intracellular signaling pathways in the pd mice may be important for maintaining bone homeostasis.     

Bone response to intermittent parathyroid hormone is altered in mice null for {beta}-Arrestin2

Intermittent PTH administration increases bone turnover, resulting in net anabolic effects on bone. These effects are primarily mediated by intracellular cAMP signaling. However, the molecular mechanisms that regulate PTH activity in bone remain incompletely understood. beta-Arrestin2, a G protein-coupled receptor regulatory protein, inhibits PTH-stimulated cAMP accumulation in vitro. Using beta-arrestin2(-/-) (KO) and wild-type (WT) mice, we investigated the response to PTH in primary osteoblasts (POB) and the effects of intermittent PTH administration on bone mass and microarchitecture in vivo. Compared with that in WT mice, PTH-stimulated intracellular cAMP was increased and sustained in KO POB. Intermittent exposure of POB to PTH significantly decreased the ratio of osteoprotegerin (OPG) receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA expression in KO POB, whereas it increased this ratio in WT POB. Total body bone mass and cortical and trabecular bone parameters were 5-10% lower in male KO mice compared with WT, and these differences were magnified upon in vivo administration of intermittent PTH (80 mug/kg.d) for 1 month. Thus, PTH significantly increased total body bone mineral content as well as vertebral trabecular bone volume and thickness in WT, but not KO mice. The anabolic response to PTH in cortical bone was also slightly more pronounced in WT than KO mice. Histomorphometry indicated that PTH prominently stimulated indexes of bone formation in both WT and KO mice, whereas it significantly increased indexes of bone resorption (i.e. osteoclast number and surface) in KO mice only. In conclusion, these results suggest that beta-arrestins may specify the activity of intermittent PTH on the skeleton by limiting PTH-induced osteoclastogenesis.     

Defective bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase

Nitric oxide (NO) is a pleiotropic signaling molecule that is produced by bone cells constitutively and in response to diverse stimuli such as proinflammatory cytokines, mechanical strain, and sex hormones. Endothelial nitric oxide synthase (eNOS) is the predominant NOS isoform expressed in bone, but its physiological role in regulating bone metabolism remains unclear. Here we studied various aspects of bone metabolism in female mice with targeted disruption of the eNOS gene. Mice with eNOS deficiency (eNOS KO) had reduced bone mineral density, and cortical thinning when compared with WT controls and histomorphometric analysis of bone revealed profound abnormalities of bone formation, with reduced osteoblast numbers, surfaces and mineral apposition rate. Studies in vitro showed that osteoblasts derived from eNOS KO mice had reduced rates of growth when compared with WT and were less well differentiated as reflected by lower levels of alkaline phosphatase activity. Mice with eNOS deficiency lost bone normally following ovariectomy but exhibited a significantly blunted anabolic response to high dose exogenous estrogen. We conclude that the eNOS pathway plays an essential role in regulating bone mass and bone turnover by modulating osteoblast function.     

Differentiation and proliferation of periosteal osteoblast progenitors are differentially regulated by estrogens and intermittent parathyroid hormone administration

The periosteum is now widely recognized as a homeostatic and therapeutic target for actions of sex steroids and intermittent PTH administration. The mechanisms by which estrogens suppress but PTH promotes periosteal expansion are not known. In this report, we show that intermittent PTH(1-34) promotes differentiation of periosteal osteoblast precursors as evidenced by the stimulation of the expression or activity of alkaline phosphatase as well as of targets of the bone morphogenetic protein 2 (BMP-2) and Wnt pathways. In contrast, 17beta-estradiol (E2) had no effect by itself. However, it attenuated PTH- or BMP-2-induced differentiation of primary periosteal osteoblast progenitors. Administration of intermittent PTH to ovariectomized mice induced rapid phosphorylation of the BMP-2 target Smad1/5/8 in the periosteum. A replacement dose of E2 had no effect by itself but suppressed PTH-induced phosphorylation of Smad1/5/8. In contrast to its effects to stimulate periosteal osteoblast differentiation, PTH promoted and subsequently suppressed proliferation of periosteal osteoblast progenitors in vitro and in vivo. E2 promoted proliferation and attenuated the antiproliferative effect of PTH. Both hormones protected periosteal osteoblasts from apoptosis induced by various proapoptotic agents. These observations suggest that the different effects of PTH and estrogens on the periosteum result from opposing actions on the recruitment of early periosteal osteoblast progenitors. Intermittent PTH promotes osteoblast differentiation from periosteum-derived mesenchymal progenitors through ERK-, BMP-, and Wnt-dependent signaling pathways. Estrogens promote proliferation of early osteoblast progenitors but inhibit their differentiation by osteogenic agents such as PTH or BMP-2.     

Activation of the inducible nitric oxide synthase pathway contributes to inflammation-induced osteoporosis by suppressing bone formation and causing osteoblast apoptosis.

OBJECTIVE: Osteoporosis is a major clinical problem in chronic inflammatory diseases such as rheumatoid arthritis. The mechanism of bone loss in this condition remains unclear, but previous studies have indicated that depressed bone formation plays a causal role. Since cytokine-induced nitric oxide (NO) production has been shown to inhibit osteoblast growth and differentiation in vitro, this study was undertaken to investigate the role of the inducible NO synthase (iNOS) pathway in the pathogenesis of inflammation-mediated osteoporosis (IMO) by studying mice with targeted inactivation of the iNOS gene (iNOS knockout [iNOS KO] mice). METHODS: IMO was induced in wild-type (WT) and iNOS KO mice by subcutaneous injections of magnesium silicate. The skeletal response was assessed at the tibial metaphysis by measurements of bone mineral density (BMD), using peripheral quantitative computed tomography, by bone histomorphometry, and by measurements of bone cell apoptosis. RESULTS: NO production increased 2.5-fold (P < 0.005) in WT mice with IMO, but did not change significantly in iNOS KO mice. Total BMD values decreased by a mean +/- SEM of 14.4+/-2.0% in WT mice with IMO, compared with a decrease of 8.6+/-1.2% in iNOS KO mice with IMO (P < 0.01). Histomorphometric analysis confirmed that trabecular bone volume was lower in WT mice with IMO compared with iNOS KO mice with IMO (16.2+/-1.5% versus 23.4+/-2.6%; P < 0.05) and showed that IMO was associated with reduced bone formation and a 320% increase in osteoblast apoptosis (P < 0.005) in WT mice. In contrast, iNOS KO mice with IMO showed less inhibition of bone formation than WT mice and showed no significant increase in osteoblast apoptosis. CONCLUSION: Inducible NOS-mediated osteoblast apoptosis and depressed bone formation play important roles in the pathogenesis of IMO.     

The immune regulatory protein B7-H3 promotes osteoblast differentiation and bone mineralization

B7-H3, a member of the B7 family of the Ig superfamily proteins, is expressed on the surface of the antigen-presenting cells and down-regulates T cell functions by engaging an unknown counterreceptor on T cells. Although B7-H3 is ubiquitously expressed, its potential nonimmune functions have not been addressed. We found that B7-H3 is highly expressed in developing bones during embryogenesis and that its expression increases as osteoblast precursor cells differentiate into mature osteoblasts. In vitro bone formation by osteoblastic cells was inhibited when B7-H3 function was interrupted by the soluble recombinant protein B7-H3-Fc. Analysis of calvarial cells derived from neonatal B7-H3 knockout (KO) mice revealed normal numbers of osteoblast precursor cells possessing a normal proliferative capacity. However, the B7-H3-deficient calvarial cells exhibited impaired osteogenic differentiation, resulting in decreased mineralized bone formation in vitro. These results suggest that B7-H3 is required for the later phase of osteoblast differentiation. Although B7-H3 KO mice had no gross skeletal abnormalities, they displayed a lower bone mineral density in cortical (but not trabecular) bones compared with WT controls. Consistent with the reduced bone mineral density, the femurs of B7-H3 KO mice were more susceptible to bone fracture compared with those of WT mice. Taken together, these results indicate that B7-H3 and its unknown counterreceptor play a positive regulatory role in bone formation. In addition, our findings identified B7-H3 as another molecule that has a dual role in the bone-immune interface.     

Regulation of beta catenin signaling and parathyroid hormone anabolic effects in bone by the matricellular protein periostin

Periostin (Postn) is a matricellular protein preferentially expressed by osteocytes and periosteal osteoblasts in response to mechanical stimulation and parathyroid hormone (PTH). Whether and how periostin expression influences bone anabolism, however, remains unknown. We investigated the skeletal response of adult Postn(-/-) and Postn(+/+) mice to intermittent PTH. Compared with Postn(+/+), Postn(-/-) mice had a lower bone mass, cortical bone volume, and strength response to PTH. PTH-stimulated bone-forming indices were all significantly lower in Postn(-/-) mice, particularly at the periosteum. Furthermore, in vivo stimulation of Wnt-β-catenin signaling by PTH, as evaluated in TOPGAL reporter mice, was inhibited in the absence of periostin (TOPGAL;Postn(-/-) mice). PTH stimulated periostin and inhibited MEF2C and sclerostin (Sost) expression in bone and osteoblasts in vitro. Recombinant periostin also suppressed Sost expression, which was mediated through the integrin αVβ3 receptor, whereas periostin-blocking antibody prevented inhibition of MEF2C and Sost by PTH. In turn, administration of a Sost-blocking antiboby partially restored the PTH-mediated increase in bone mass in Postn(-/-) mice. In addition, primary osteoblasts from Postn(-/-) mice showed a lower proliferation, mineralization, and migration, both spontaneously and in response to PTH. Osteoblastic gene expression levels confirmed a defect of Postn(-/-) osteoblast differentiation with and without PTH, as well as an increased osteoblast apoptosis in the absence of periostin. These data elucidate the complex role of periostin on bone anabolism, through the regulation of Sost, Wnt-β-catenin signaling, and osteoblast differentiation.     

Activation of the inducible nitric oxide synthase pathway contributes to inflammation‐induced osteoporosis by suppressing bone formation and causing osteoblast apoptosis

Objective

Osteoporosis is a major clinical problem in chronic inflammatory diseases such as rheumatoid arthritis. The mechanism of bone loss in this condition remains unclear, but previous studies have indicated that depressed bone formation plays a causal role. Since cytokine‐induced nitric oxide (NO) production has been shown to inhibit osteoblast growth and differentiation in vitro, this study was undertaken to investigate the role of the inducible NO synthase (iNOS) pathway in the pathogenesis of inflammation‐mediated osteoporosis (IMO) by studying mice with targeted inactivation of the iNOS gene (iNOS knockout [iNOS KO] mice).

Methods

IMO was induced in wild‐type (WT) and iNOS KO mice by subcutaneous injections of magnesium silicate. The skeletal response was assessed at the tibial metaphysis by measurements of bone mineral density (BMD), using peripheral quantitative computed tomography, by bone histomorphometry, and by measurements of bone cell apoptosis.

Results

NO production increased 2.5‐fold (P < 0.005) in WT mice with IMO, but did not change significantly in iNOS KO mice. Total BMD values decreased by a mean ± SEM of 14.4 ± 2.0% in WT mice with IMO, compared with a decrease of 8.6 ± 1.2% in iNOS KO mice with IMO (P < 0.01). Histomorphometric analysis confirmed that trabecular bone volume was lower in WT mice with IMO compared with iNOS KO mice with IMO (16.2 ± 1.5% versus 23.4 ± 2.6%; P < 0.05) and showed that IMO was associated with reduced bone formation and a 320% increase in osteoblast apoptosis (P < 0.005) in WT mice. In contrast, iNOS KO mice with IMO showed less inhibition of bone formation than WT mice and showed no significant increase in osteoblast apoptosis.

Conclusion

Inducible NOS–mediated osteoblast apoptosis and depressed bone formation play important roles in the pathogenesis of IMO.

     

Rapid modulation of osteoblast ion channel responses by 1alpha,25(OH)2-vitamin D3 requires the presence of a functional vitamin D nuclear receptor

1alpha,25(OH)(2)-Vitamin D(3) (1,25D) modulates osteoblast gene expression of bone matrix proteins via a nuclear vitamin D receptor (VDR) and also modifies the electrical state of the plasma membrane through rapid nongenomic mechanisms still not fully understood. The physiological significance of 1,25D membrane-initiated effects remains unclear. To elucidate whether the VDR is required for 1,25D-promoted electrical responses, we studied 1,25D modulation of ion channel activities in calvarial osteoblasts isolated from VDR knockout (KO) and WT mice. At depolarizing potentials, Cl(-) currents were significantly potentiated (13.5 +/- 1.6-fold increase, n = 12) by 5 nM 1,25D in VDR WT but not in KO (0.96 +/- 0.3 fold increase, n = 11) osteoblasts. L-type Ca(2+) currents significantly shift their peak activation by -9.3 +/- 0.7 mV (n = 10) in the presence of 5 nM 1,25D in VDR WT but not in KO cells, thus facilitating Ca(2+) influx. Furthermore, we found that 1,25D significantly increased whole-cell capacitance in VDR WT (DeltaCap = 2.3 +/- 0.4 pF, n = 8) but not in KO osteoblasts (DeltaCap = 0.3 +/- 0.1 pF, n = 8); this corresponds to a rapid (1-2 min) fusion in WT of 71 +/- 33 versus in KO only 9 +/- 6 individual secretory granules. We conclude that, in calvarial osteoblasts, 1,25D modulates ion channel activities only in cells with a functional VDR and that this effect is coupled to exocytosis. This is a demonstration of the requirement of a functional classic steroid receptor for the rapid hormonal modulation of electric currents linked to secretory activities in a target cell.     

Lack of insulin-like growth factor I exaggerates the effect of calcium deficiency on bone accretion in mice

Recent studies provide evidence that the GH/IGF-I axis plays a critical role in the regulation of bone accretion that occurs during puberty and that the peak bone mineral density (BMD) is dependent on the amount of dietary calcium intake during the active growth phases. To evaluate whether IGF-I deficiency exaggerates the effect of calcium deficiency on bone accretion during active growth phases, IGF-I knockout (KO) and wild-type (WT) mice were fed with low calcium (0.01%) or normal calcium (0.6%) for 2 wk during the pubertal growth phase and were labeled with tetracycline. The low calcium diet caused significant decreases in endosteal bone formation parameters and a much greater increase in the resorbing surface of both the endosteum and periosteum of the tibia of IGF-I KO mice compared with WT mice. Accordingly, femur BMD measured by dual energy x-ray absorptiometry or peripheral quantitative computed tomography increased significantly in IGF-I WT mice fed the low calcium diet, but not in IGF-I KO mice. IGF-I-deficient mice fed the normal calcium diet showed elevated PTH levels, decreased serum 1,25-dihydroxyvitamin D and serum calcium levels at baseline. Serum calcium changes due to calcium deficiency were greater in IGF-I KO mice compared with WT mice. PTH levels were 7-fold higher in IGF-I KO mice fed normal calcium compared with WT mice, which was further elevated in mice fed the low calcium diet. Treatment of IGF-I-deficient lit/lit mice with GH decreased the serum PTH level by 70% (P < 0.01). Based on these and past findings, we conclude that: 1) IGF-I deficiency exaggerates the negative effects of calcium deficiency on bone accretion; and 2) IGF-I deficiency may lead to 1,25-dihydroxyvitamin D deficiency and elevated PTH levels even under normal calcium diet.     

Osteonectin-null mutation compromises osteoblast formation,maturation, and survival

Osteonectin, also known as SPARC (secreted protein acidic and rich in cysteine) or BM-40, is one of the most abundant noncollagenous proteins in bone. Analysis of osteonectin-null mice revealed that osteonectin is necessary for the maintenance of bone mass and normal remodeling, as osteonectin-null mice have decreased osteoblast number and bone formation rate. Cultures of bone marrow stromal cells and osteoblasts from control and osteonectin-null mice were used to determine the cellular basis for the mutant phenotype. We found that marrow stroma from osteonectin-null mice contains fewer osteoblastic precursors than that of control mice, and the osteonectin-null mutation did not affect the proliferation rate of stromal cells or osteoblasts. Whereas osteonectin-null cells could adopt an osteoblastic phenotype, a smaller proportion of these cells expressed markers of a fully differentiated osteoblast. Mutant cells exhibited decreased formation of mineralized nodules, as well as diminished expression of osteocalcin mRNA and response to PTH. Furthermore, osteonectin-null cells showed an increased tendency to form adipocytes, with enhanced expression of the adipocytic markers adipsin and CCAAT/enhancer binding protein delta. Osteonectin-null cells were also more susceptible to environmental stresses. These data indicate that osteonectin is important for osteoblast formation, maturation, and survival.     

Gp130-mediated signaling is necessary for normal osteoblastic function in vivo and in vitro

Previous studies have shown that mice missing gp130, the common receptor subunit for many cytokines, die at or before birth with multiple skeletal abnormalities. Furthermore, interactions between PTH and gp130 signaling have suggested that gp130 signaling might influence calcium homeostasis. We, therefore, examined the function of osteoblasts, osteoclasts, and calcium homeostasis in gp130(-/-) mice, both in vivo and in vitro. Osteoblasts from these mice exhibit widespread abnormalities, including decreased alkaline phosphatase mRNA and protein, both in vivo and in osteoblast cultures. Although osteoclast number is increased in gp130(-/-) fetuses, these osteoclasts exhibit abnormalities in the resorptive organelle and the ruffled border, and the mice are mildly hypocalcemic. Although the hypocalcemia is associated with secondary hyperparathyroidism, the increase in PTH does not explain the increase in osteoclast number because removal of the PTH gene in gp130(-/-) fetuses does not importantly change osteoclast number. Calvarial bone resorption in response to PTH is defective, as is the ability of osteoblastic cells from gp130(-/-) mice to stimulate osteoclastogenesis from normal precursors in vitro or to increase receptor activator of nuclear factor-kappa B ligand mRNA levels after exposure to PTH. These studies demonstrate the importance of gp130 signaling for osteoblast function and calcium homeostasis.     

Response to parathyroid hormone and 1,25-dihydroxyvitamin D3 of bone-derived cells isolated from normal children and children with abnormalities in skeletal development

In order to evaluate the role of intrinsic defects in osteoblast function in the pathogenesis of diseases of skeletal development, we developed techniques which permit the evaluation of the metabolic properties of bone-derived cells in vitro. Cells from control children demonstrated a variety of properties classically attributed to osteoblasts (presence of alkaline phosphatase positive cells and synthesis of bone gla protein) and responded to PTH (cAMP production) and to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ([3H]25-hydroxyvitamin D3 conversion into [3H]24,25-dihydroxyvitamin D3 and bone gla protein secretion). Using these techniques we evaluated the function of cultured bone cells from patients with three rare diseases of skeletal development. Cells from a patient with rickets resistant to 1,25(OH)2D3 were resistant to 1,25(OH)2D3 but responded normally to PTH. Cells from a patient with acroosteolysis with osteoporosis responded normally to PTH and 1,25(OH)2D3. Cells from a patient with hyperphosphatasia with osteoectasia responded normally to 1,25(OH)2D3 but did not respond to PTH. The results demonstrate that bone cell cultures can provide information about the role of osteoblast dysfunction in such diseases.     

aP2-Cre-mediated inactivation of acetyl-CoA carboxylase 1 causes growth retardation and reduced lipid accumulation in adipose tissues

Adipose tissue is one of the major sites for fatty acid synthesis and lipid storage. We generated adipose (fat)-specific ACC1 knockout (FACC1KO) mice using the aP2-Cre/loxP system. FACC1KO mice showed prenatal growth retardation; after weaning, however, their weight gain was comparable to that of wild-type (WT) mice on a normal diet. Under lipogenic conditions of fasting/re-feeding a fat-free diet, lipid accumulation in adipose tissues of FACC1KO mice was significantly decreased; this is consistent with a 50–66% reduction in the ACC activity in these tissues compared with that of WT mice. Surprisingly, FACC1KO mice manifested skeletal growth retardation phenotype accompanied by decreased chondrocyte proliferation in the growth plate and lower trabecular bone density. In addition, there was about a 30% decrease in serum insulin-like growth factor I (IGF1), and while the serum leptin level was decreased by about 50%, it did not counteract the osteopenic effects of IGF1 on the bone. Fatty acid analyses of mutant bone lipids revealed relatively higher levels of C18:2n-6 and C18:3n-3 and lower levels of their elongation C20 homologs than that of WT cohorts, leading to lower levels of C20 homologs and bone development. Moreover, aP2-Cre-mediated ACC1 inactivation in bone tissue led to a decreased number of osteoblasts but not of osteoclasts. The downregulation of ACC1 on osteoblastogenesis may be the cause for the osteopenia phenotype of FACC1KO bone homeostasis.     

Do cyclooxygenase-2 knockout mice have primary hyperparathyroidism?

The absence of cyclooxygenase-2 (COX-2) activity in vitro reduces differentiation of both bone-forming and bone-resorbing cells. To examine the balance of COX-2 effects on bone in vivo, we studied COX-2 knockout (KO) and wild-type (WT) mice. After weaning, KO mice died 4 times faster than WT mice, consistent with reports of progressive renal failure in KO mice. Among KO mice killed at 4 months of age, some had renal failure with marked secondary hyperparathyroidism, but others appeared healthy. On the assumption that renal failure was not inevitable in COX-2 KO mice and that phenotypic differences might increase with age, we studied KO mice surviving to 10 months of age with serum creatinine levels similar to those of WT mice. In 10-month-old male KO mice, serum calcium and PTH, but not phosphorus, levels were increased compared with those in WT mice. 1,25-Dihydroxyvitamin D(3) levels were markedly elevated in KO mice. Skeletal analysis showed small nonsignificant decreases in cortical bone density by BMD and either an increase (distal femur, by microcomputed tomography) or no difference (distal femur, by static histomorphometry) in trabecular bone density in KO mice. There was a trend toward increased percent osteoblastic and osteoclastic surfaces, and on dynamic histomorphometry, the rates of trabecular bone formation and mineral apposition were increased in KO mice relative to WT mice. Similar trends were observed for most parameters in 10-month-old female COX-2 KO mice. However, rates of trabecular bone formation and mineral apposition were increased in 10-month-old WT females compared with males and did not increase further in female KO mice. These data suggest that COX-2 KO mice with intact renal function have primary hyperparathyroidism, and that effects of increased PTH and 1,25-dihydroxyvitamin D(3) to increase bone turnover may compensate for the absence of COX-2.     

Galectin-3 is essential for proper bone cell differentiation and activity,bone remodeling and biomechanical competence in mice

ObjectiveGalectin-3 is constitutively expressed in bone cells and was recently shown to modulate osteogenic transdifferentiation of vascular smooth muscle cells and atherosclerotic calcification. However, the role of galectin-3 in bone physiology is largely undefined. To address this issue, we analyzed (1) the skeletal features of 1-, 3- and 6-month-old galectin-3 null (Lgals3^−/−) and wild type (WT) mice and (2) the differentiation and function of osteoblasts and osteoclasts derived from these animals.MethodsLong bone phenotype, gene expression profile, and remodeling were investigated by micro-computed tomography, real time-PCR, static and dynamic histomorphometry, and assessment of biochemical markers of bone resorption and formation. Bone competence was also evaluated by biomechanical testing at 3 months. In vitro, the effects of galectin-3 deficiency on bone cell differentiation and function were investigated by assessing (a) gene expression of osteoblast markers, alkaline phosphatase activity, mineralization assay, and WNT/β-catenin signaling (of which galectin-3 is a known regulator) in osteoblasts; and (b) tartrate-resistant acid phosphatase activity and bone resorption activity in osteoclasts.ResultsLgals3^−/− mice revealed a wide range of age-dependent alterations including lower bone formation and higher bone resorption, accelerated age-dependent trabecular bone loss (p < 0.01 vs. WT at 3 months) and reduced bone strength (p < 0.01 vs. WT at 3 months). These abnormalities were accompanied by a steady inflammatory state, as revealed by higher bone expression of the pro-inflammatory cytokines interleukin (IL)-1β and IL-6 (p < 0.001 vs. WT at 3 months), increased content of osteal macrophages (p < 0.01 vs. WT at 3 months), and reduced expression of markers of alternative (M2) macrophage activation. Lgals3^−/− osteoblasts and osteoclasts showed impaired terminal differentiation, reduced mineralization capacity (p < 0.01 vs. WT cells) and resorption activity (p < 0.01 vs. WT cells). Mechanistically, impaired differentiation and function of Lgals3^−/− osteoblasts was associated with altered WNT/β-catenin signaling (p < 0.01 vs. WT cells).ConclusionsThese data provide evidence for a contribution of galectin-3 to bone cell maturation and function, bone remodeling, and biomechanical competence, thus identifying galectin-3 as a promising therapeutic target for age-related disorders of bone remodeling.     

Calcineurin regulates bone formation by the osteoblast

Two of the most commonly used immunosuppressants, cyclosporine A and tacrolimus (FK506), inhibit the activity of a ubiquitously expressed Ca(2+)/calmodulin-sensitive phosphatase, calcineurin. Because both drugs also cause profound bone loss in humans and in animal models, we explored whether calcineurin played a role in regulating skeletal remodeling. We found that osteoblasts contained mRNA and protein for all isoforms of calcineurin A and B. TAT-assisted transduction of fusion protein TAT-calcineurin Aalpha into osteoblasts resulted in the enhanced expression of the osteoblast differentiation markers Runx-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. This expression was associated with a dramatic enhancement of bone formation in intact calvarial cultures. Calcineurin Aalpha(-/-) mice displayed severe osteoporosis, markedly reduced mineral apposition rates, and attenuated colony formation in 10-day ex vivo stromal cell cultures. The latter was associated with significant reductions in Runx2, bone sialoprotein, and osteocalcin expression, paralleled by similar decreases in response to FK506. Together, the gain- and loss-of-function experiments indicate that calcineurin regulates bone formation through an effect on osteoblast differentiation.     

Effects of loss of steroid receptor coactivator-1 on the skeletal response to estrogen in mice

Steroid receptor coactivator (SRC)-1 is an important nuclear receptor coactivator that enhances estrogen (E) action in many tissues, but its role in mediating E effects on bone is unknown. Thus, we assessed the skeletal response to ovariectomy (ovx) and E replacement in SRC-1 knockout (KO) mice compared with wild-type (WT) littermates. Bone mineral density was measured by dual-energy x-ray absorptiometry and peripheral quantitative computed tomography at baseline and after 2 months of sham surgery, ovx, or ovx plus E replacement. Microcomputed tomography and bone histomorphometry were also performed at the end of the study. Both WT and SRC-1 KO mice lost bone at multiple sites after ovx; however, although an estradiol (E(2)) dose of 10 microg/kg.d completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis) in the WT mice, it was entirely ineffective in preventing cancellous bone loss at these sites in the SRC-1 KO mice. This E(2) dose was, however, equally effective on cortical bone in the tibia in the SRC-1 KO and WT mice. Moreover, a 4-fold higher dose of E(2) was able to overcome the deficit in E action in cancellous bone in the SRC-1 KO mice. These findings establish that, in mice, loss of SRC-1 leads to skeletal resistance to E predominantly in cancellous bone.     

Conditional expression of a Gi-coupled receptor in osteoblasts results in trabecular osteopenia

G protein-coupled receptors (GPCRs) coupled to activation of Gs, such as the PTH1 receptor (PTH1R), have long been known to regulate skeletal function and homeostasis. However, the role of GPCRs coupled to other G proteins such as Gi is not well established. We used the tet-off system to regulate the expression of an activated Gi-coupled GPCR (Ro1) in osteoblasts in vivo. Skeletal phenotypes were assessed in mice expressing Ro1 from conception, from late stages of embryogenesis, and after weaning. Long bones were assessed histologically and by microcomputed tomography. Expression of Ro1 from conception resulted in neonatal lethality that was associated with reduced bone mineralization. Expression of Ro1 starting at late embryogenesis resulted in a severe trabecular bone deficit at 12 wk of age (>51% reduction in trabecular bone volume fraction in the proximal tibia compared with sex-matched control littermates; n = 11; P < 0.01). Ro1 expression for 8 wk beginning at 4 wk of age resulted in a more than 20% reduction in trabecular bone volume fraction compared with sex-matched control littermates (n = 16; P < 0.01). Bone histomorphometry revealed that Ro1 expression is associated with reduced rates of bone formation and mineral apposition without a significant change in osteoblast or osteoclast surface. Our results indicate that signaling by a Gi-coupled GPCR in osteoblasts leads to osteopenia resulting from a reduction in trabecular bone formation. The severity of the phenotype is related to the timing and duration of Ro1 expression during growth and development. The skeletal phenotype in Ro1 mice bears some similarity to that produced by knockout of Gs-alpha expression in osteoblasts and thus may be due at least in part to Gi-mediated inhibition of adenylyl cyclase.