Corticosterone selectively targets endo-cortical surfaces by an osteoblast-dependent mechanism.

BACKGROUND: The pathogenesis of glucocorticoid-induced osteoporosis remains ill defined. In this study, we examined the role of the osteoblast in mediating the effects of exogenous glucocorticoids on cortical and trabecular bone, employing the Col2.3-11ßHSD2 transgenic mouse model of osteoblast-targeted disruption of glucocorticoid signalling. METHODS: Eight week-old male transgenic (tg) and wild-type (WT) mice (n=20-23/group) were treated with either 1.5 mg corticosterone (CS) or placebo for 4 weeks. Serum tartrate-resistant acid phosphatase 5b (TRAP5b) and osteocalcin (OCN) were measured throughout the study. Tibiae and lumbar vertebrae were analysed by micro-CT and histomorphometry at endpoint. RESULTS: CS suppressed serum OCN levels in WT and tg mice, although they remained higher in tg animals at all time points (p<0.05). Serum TRAP5b levels increased in WT mice only. The effect of CS on cortical bone differed by site: At the endosteal surface, exposure to CS significantly increased bone resorption and reduced bone formation, resulting in a larger bone marrow cavity cross-sectional area (p<0.01). In contrast, at the pericortical surface bone resorption was significantly decreased accompanied with a significant increase in pericortical cross-sectional area (p<0.05) while bone formation remained unaffected. Vertebral cortical thickness and area were reduced in CS treatment mice. Tg mice were partially protected from the effects of exogenous CS, both on a cellular and structural level. At the CS doses used in this study, trabecular bone remained largely unaffected. CONCLUSION: Endocortical osteoblasts appear to be particularly sensitive to the detrimental actions of exogenous glucocorticoids. The increase in tibial pericortical cross-sectional area and the according changes in pericortical circumference suggest an anabolic bone response to GC treatment at this site. The protection of tg mice from these effects indicates that both catabolic and anabolic action of glucocorticoids are, at least in part, mediated by osteoblasts.

Follicle-stimulating hormone increases bone mass in female mice.

Elevated follicle-stimulating hormone (FSH) activity is proposed to directly cause bone loss independent of estradiol deficiency in aging women. Using transgenic female mice expressing human FSH (TgFSH), we now reveal that TgFSH dose-dependently increased bone mass, markedly elevating tibial and vertebral trabecular bone volume. Furthermore, TgFSH stimulated a striking accrual of bone mass in hypogonadal mice lacking endogenous FSH and luteinizing hormone (LH) function, showing that FSH-induced bone mass occurred independently of background LH or estradiol levels. Higher TgFSH levels increased osteoblast surfaces in trabecular bone and stimulated de novo bone formation, filling marrow spaces with woven rather than lamellar bone, reflective of a strong anabolic stimulus. Trabecular bone volume correlated positively with ovarian-derived serum inhibin A or testosterone levels in TgFSH mice, and ovariectomy abolished TgFSH-induced bone formation, proving that FSH effects on bone require an ovary-dependent pathway. No detectable FSH receptor mRNA in mouse bone or cultured osteoblasts or osteoclasts indicated that FSH did not directly stimulate bone. Therefore, contrary to proposed FSH-induced bone loss, our findings demonstrate that FSH has dose-dependent anabolic effects on bone via an ovary-dependent mechanism, which is independent of LH activity, and does not involve direct FSH actions on bone cells.

Genetic and hormonal control of bone volume, architecture, and remodeling in XXY mice.

Klinefelter syndrome is the most common chromosomal aneuploidy in men (XXY karyotype, 1 in 600 live births) and results in testicular (infertility and androgen deficiency) and nontesticular (cognitive impairment and osteoporosis) deficits. The extent to which skeletal changes are due to testosterone deficiency or arise directly from gene overdosage cannot be determined easily in humans. To answer this, we generated XXY mice through a four-generation breeding scheme. Eight intact XXY and 9 XY littermate controls and 8 castrated XXY mice and 8 castrated XY littermate controls were euthanized at 1 year of age. Castration occurred 6 months prior to killing. A third group of 9 XXY and 11 XY littermates were castrated and simultaneously implanted with a 1-cm Silastic testosterone capsule 8 weeks prior to sacrifice. Tibias were harvested from all three groups and examined by micro-computed tomography and histomorphometry. Blood testosterone concentration was assayed by radioimmunoassay. Compared with intact XY controls, intact androgen-deficient XXY mice had lower bone volume (6.8% +/- 1.2% versus 8.8% +/- 1.7%, mean +/- SD, p = .01) and thinner trabeculae (50 +/- 4 µm versus 57 +/- 5 µm, p = .007). Trabecular separation (270 +/- 20 µm versus 270 +/- 20 µm) or osteoclast number relative to bone surface (2.4 +/- 1.0/mm2 versus 2.7 +/- 1.5/mm2) did not differ significantly. Testosterone-replaced XXY mice continued to show lower bone volume (5.5% +/- 2.4% versus 8.1% +/- 3.5%, p = .026). They also exhibited greater trabecular separation (380 +/- 69 µm versus 324 +/- 62 µm, p = .040) but equivalent blood testosterone concentrations (6.3 +/- 1.8 ng/mL versus 8.2 +/- 4.2 ng/mL, p = .28) compared with testosterone-replaced XY littermates. In contrast, castration alone drastically decreased bone volume (p < .001), trabecular thickness (p = .05), and trabecular separation (p < .01) to such a great extent that differences between XXY and XY mice were undetectable. In conclusion, XXY mice replicate many features of human Klinefelter syndrome and therefore are a useful model for studying bone. Testosterone deficiency does not explain the bone phenotype because testosterone-replaced XXY mice show reduced bone volume despite similar blood testosterone levels.

Vitamin D deficiency promotes human breast cancer growth in a murine model of bone metastasis.

Vitamin D exerts antiproliferative, prodifferentiation, and proapoptotic effects on nonclassic target tissues such as breast. Blood levels of 25-hydroxyvitamin D [25(OH)D], the most sensitive indicator of vitamin D status, are inversely correlated with breast cancer risk; however, a causal relationship between vitamin D deficiency and breast cancer growth in bone has not been assessed. We examined the effect of vitamin D deficiency on the intraskeletal growth of the human breast cancer cell line MDA-MB-231-TxSA in a murine model of malignant bone lesions. Subsets of mice were treated concurrently with osteoprotegerin (OPG) to abrogate bone resorption. Outcomes were assessed by repeated radiographic and end-point micro-computed tomography and histologic analyses. Mice weaned onto a vitamin D-free diet developed vitamin D deficiency within 4 weeks [mean +/- SE serum 25(OH)D: 11.5 +/- 0.5 nmol/L], which was sustained throughout the study and was associated with secondary hyperparathyroidism and accelerated bone turnover. Osteolytic lesions appeared earlier and were significantly larger in vitamin D-deficient than in vitamin D-sufficient mice after 2 weeks (radiographic osteolysis: +121.5%; histologic tumor area: +314%; P < 0.05). Although OPG treatment reduced the size of radiographic osteolyses and tumor area in both groups, tumors remained larger in OPG-treated vitamin D-deficient compared with OPG-treated vitamin D-sufficient mice (0.53 +/- 0.05 mm(2) versus 0.19 +/- 0.05 mm2; P < 0.05). We conclude that vitamin D deficiency promotes the growth of human breast cancer cells in the bones of nude mice. These effects are partly mediated through secondary changes in the bone microenvironment, along with direct effects of vitamin D on tumor growth.

Osteoblast-targeted disruption of glucocorticoid signalling does not delay intramembranous bone healing.

OBJECTIVE: Glucocorticoids at pharmacological doses have been shown to interfere with fracture repair. The role of endogenous glucocorticoids in fracture healing is not well understood. We examined whether endogenous glucocorticoids affect bone healing in an in vivo model of cortical defect repair. METHODS: Experiments were performed using a well characterised mouse model in which intracellular glucocorticoid signalling was disrupted in osteoblasts through transgenic overexpression of 11beta-hydroxysteroid-dehydrogenase type 2 (11beta-HSD2) under the control of a collagen type I promoter (Col2.3-11beta-HSD2). Unicortical bone defects (ø 0.8mm) were created in the tibiae of 7-week-old male transgenic mice and their wild-type littermates. Repair was assessed via histomorphometry, immunohistochemistry and microcomputed tomography (micro-CT) analysis at 1-3 weeks after defect creation. RESULTS: At week 1, micro-CT images of the defect demonstrated formation of mineralized intramembranous bone which increased in volume and density by week 2. At week 3, healing of the defect was nearly complete in all animals. Analysis by histomorphometry and micro-CT revealed that repair of the bony defect was similar in Col2.3-11beta-HSD2 transgenic animals and their wild-type littermates at all time-points. CONCLUSION: Disrupting endogenous glucocorticoid signalling in mature osteoblasts did not affect intramembranous fracture healing in a tibia defect repair model. It remains to be shown whether glucocorticoid signalling has a role in endochondral fracture healing.

Transgenic disruption of glucocorticoid signaling in mature osteoblasts and osteocytes attenuates K/BxN mouse serum-induced arthritis in vivo.

OBJECTIVE: Endogenous glucocorticoids (GCs) modulate numerous biologic systems involved in the initiation and maintenance of arthritis. Bone cells play a critical role in the progression of arthritis, and some of the effects of GCs on inflammation may be mediated via these cells. The aim of this study was to investigate the impact of osteoblast-targeted disruption of GC signaling on joint inflammation, cartilage damage, and bone metabolism in the K/BxN mouse serum transfer model of autoimmune arthritis. METHODS: Intracellular GC signaling was disrupted in osteoblasts through transgenic overexpression of 11beta-hydroxysteroid dehydrogenase type 2 under the control of a type I collagen promoter. Arthritis was induced in 5-week-old male transgenic mice and their wild-type (WT) littermates, and paw swelling was assessed daily until the mice were killed. The mice were examined by histology, histomorphometry, and microfocal computed tomography, and serum was analyzed for cytokines, adrenocorticotropic hormone, and corticosterone. RESULTS: Acute arthritis developed in both transgenic and WT mice treated with K/BxN mouse serum. However, the arthritis and local inflammatory activity were significantly attenuated in transgenic mice, as judged by clinical and histologic indices of inflammation and cartilage damage. Bone turnover and bone volume remained unchanged in arthritic transgenic mice, while WT mice exhibited stimulated bone resorption, suppressed osteoblast activity, and significantly reduced bone volume, compatible with the known effects of active inflammation on bone. Circulating levels of proinflammatory cytokines tended to be lower in arthritic transgenic mice than in control transgenic mice. CONCLUSION: Disruption of GC signaling in osteoblasts significantly attenuates K/BxN mouse serum-induced autoimmune arthritis in mice. These data suggest that osteoblasts modulate the immune-mediated inflammatory response via a GC-dependent pathway.

Endogenous glucocorticoid signalling in osteoblasts is necessary to maintain normal bone structure in mice.

The role of endogenous glucocorticosteroids (GC) in bone development is ill-defined. Using the Col2.3-11betaHSD2 transgenic (tg) mouse model, we examined the effect of osteoblast-targeted disruption of intracellular GC signalling on bone growth and strength, and its modulation by factors such as age, gender and skeletal site. Tibiae and L3 vertebrae of 3 and 7-week-old, male and female wild type (WT) mice and their tg, age and sex matched littermates were analysed by micro-CT and mechanical testing. Data were analysed separately for 3 and 7-week-old mice by 2-way ANOVA using genotype (WT, tg), gender and their interactions as factors. Transgenic mice were characterised by lower bone volume, lower trabecular number and higher trabecular separation in tibial trabecular bone, as well as lower tibial cortical bone area and periosteal and endosteal perimeters. These changes resulted in a marked decrease in mechanical bone strength and stiffness in sexually mature, 7-week-old mice. In the tibia, the observed transgene effect was present in 3 and 7-week-old animals, indicating that the biological effect of disrupted GC signalling was independent of sexual maturity. This was not the case for the vertebral bones, where significant differences between tg and WT mice were seen in 7 but not in 3-week-old animals, suggesting that the effects of the transgene at this site may be modulated by age and/or changes in circulating sex hormone levels. Taken together, our results demonstrate that endogenous glucocorticoids may be required for normal bone growth but that their effect on bone structure and strength varies according to the skeletal site and sexual maturity of the animals.

Glucocorticoid-dependent Wnt signaling by mature osteoblasts is a key regulator of cranial skeletal development in mice.

Glucocorticoids are important regulators of bone cell differentiation and mesenchymal lineage commitment. Using a cell-specific approach of osteoblast-targeted transgenic disruption of intracellular glucocorticoid signaling, we discovered a novel molecular pathway by which glucocorticoids, mainly through the mature osteoblast, regulate the cellular mechanisms that govern cranial skeleton development. Embryonic and neonatal transgenic mice revealed a distinct phenotype characterized by hypoplasia and osteopenia of the cranial skeleton; disorganized frontal, parietal and interparietal bones; increased suture patency; ectopic differentiation of cartilage in the sagittal suture; and disturbed postnatal removal of parietal cartilage. Concurrently, expression of Mmp14, an enzyme essential for calvarial cartilage removal, was markedly reduced in parietal bone and cartilage of transgenic animals. Expression of Wnt9a and Wnt10b was significantly reduced in osteoblasts with disrupted glucocorticoid signaling, and accumulation of beta-catenin, the upstream regulator of Mmp14 expression, was decreased in osteoblasts, chondrocytes and mesenchymal progenitors of transgenic mice. Supracalvarial injection of Wnt3a protein rescued the transgenic cranial phenotype. These results define novel roles for glucocorticoids in skeletal development and delineate how osteoblasts--under steroid hormone control--orchestrate the intricate process of intramembranous bone formation by directing mesenchymal cell commitment towards osteoblastic differentiation while simultaneously initiating and controlling cartilage dissolution in the postnatal mouse.

The challenge of continuous exogenous glucocorticoid administration in mice.

BACKGROUND: Chronic administration of exogenous glucocorticoids is often required in experimental research. We compared the efficacy and reliability of three different methods of continuous glucocorticoid administration in mice. MATERIALS AND METHODS: Male CD1 Swiss White mice aged 7-9 weeks received corticosterone (CS) or carrier by either subcutaneous (s.c.) injection (n=15), s.c. implantation of micro-osmotic pumps (n=20) or s.c. implantation of slow-release pellets (n=20). Serial blood samples were taken for the measurement of plasma CS and osteocalcin (OC). Bone structural parameters were analysed by micro-computed tomography (micro-CT) in animals treated via slow-release pellets for 4 weeks. RESULTS: Injection of CS (10 mg/kg) resulted in peak plasma CS levels of up to 2600 microg/L after 1 h, with levels returning to baseline within 4 h post-injection. Micro-osmotic pumps failed to consistently alter plasma CS levels and had variable effects on plasma OC levels. Implantation of 10 mg CS pellets induced hypercorticosteronemia within 24 h but levels returned to baseline within 7 days. Plasma OC levels fell rapidly on day 1 and remained suppressed until day 7. Weekly replacement of pellets maintained elevated plasma CS and suppressed plasma OC concentrations, and resulted in significant bone loss at the tibia and spine after 28 days. CONCLUSION: Once-weekly s.c. implantation of slow-release pellets to mice appears to result in relatively consistent plasma CS and OC levels with significant biological effects. However, at least in our hands, no method delivered CS at a constant rate and variability in plasma CS levels was pronounced.

Accelerated bone resorption, due to dietary calcium deficiency, promotes breast cancer tumor growth in bone.

The skeleton is a major site of breast cancer metastases. High bone turnover increases risk of disease progression and death. However, there is no direct evidence that high bone turnover is causally associated with the establishment and progression of metastases. In this study, we investigate the effects of high bone turnover in a model of breast cancer growth in bone. Female nude mice commenced a diet containing normal (0.6%; 'Normal-Ca') or low (0.1%; 'Low-Ca') calcium content. Mice were concurrently treated with vehicle or osteoprotegerin (1 mg/kg/d s.c; n = 16 per group). Three days later (day 0), 50,000 Tx-SA cells (variant of MDA-MB-231 cells) were implanted by intratibial injection. On day 0, mice receiving Low-Ca had increased serum parathyroid hormone (PTH) and tartrate-resistant acid phosphatase 5b levels, indicating secondary hyperparathyroidism and high bone turnover, which was maintained until day 17. Osteoprotegerin increased serum PTH but profoundly reduced bone resorption. On day 17, in mice receiving Low-Ca alone, lytic lesion area, tumor area, and cancer cell proliferation increased by 43%, 24%, and 24%, respectively, compared with mice receiving Normal Ca (P < 0.01). Osteoprotegerin treatment completely inhibited lytic lesions, reduced tumor area, decreased cancer cell proliferation, and increased cancer cell apoptosis. Increased bone turnover, due to dietary calcium deficiency, promotes tumor growth in bone, independent of the action of PTH. Breast cancer patients frequently have low dietary calcium intake and high bone turnover. Treatment to correct calcium insufficiency and/or treatment with antiresorptive agents, such as osteoprotegerin, may be of benefit in the adjuvant as well as palliative setting.

Association analysis of the polymorphisms of the VDR gene with bone mineral density and the occurrence of fractures.

Associations of the FokI, BsmI, ApaI, and TaqI polymorphisms of the vitamin D receptor (VDR) gene with the bone mineral density (BMD) of the lumbar part of the spinal column (BMD LS) and the neck of the femur (BMD FN), and with the occurrence of fractures, were studied using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis on DNA isolated from peripheral blood of 239 women and 40 men from the region of western Poland. Three polymorphisms of the 3' end of the VDR gene (BsmI, ApaI, TaqI) indicated a strong linkage disequilibrium. Association analysis of the VDR gene FokI polymorphism with BMD LS showed a dose effect of allele f. The association of the bAT haplotype of the BsmI, ApaI, and TaqI polymorphisms of the VDR gene with BMD FN was statistically significant. The association of the ApaI polymorphism with the occurrence of fractures was observed. Associations were also observed between the occurrence of fractures and the baT haplotypes of the VDR gene.

Polymorphism of VDR gene--the most effective molecular marker of osteoporotic bone fractures risk within postmenopausal women from Wielkopolska region of Poland.

The major public health problem which will arise is a frequency of osteoporosis. The first manifestations of this disease are often bone fractures. Identification and evaluation of individual bone fracture risk will be the most effective way of solving the problem. Genetic determination of osteoporosis is unquestionable. The aim of this study is to detect which variants of genotypes lead to illness. We investigated 187 patients with osteoporosis (161 women, 26 men) and 19 healthy subjects. Polymorphisms of the following genes were investigated: OPG, VDR, ESR1, TGFB1 COL1A1, and BMP2. The statistically significant relationship between BMD value and T allele of Taq I VDR gene were found. Genotypes: aa, bb, TT of VDR gene occur more frequently in polish osteoporotic population in Wielkopolska region within patients with higher risk of bone fractures.

[Does smoking influence the course of Crohn's disease in patients with the presence of gene NOD2/CARD15 mutation?]. / Czy palenie papierosów ma wplyw na przebieg choroby Lesniowskiego-Crohna u chorych z obecnym wariantem mutacji genu NOD2/CARD15?

Inflammatory bowel diseases can be divided into two diseases: ulcerative colitis and Crohn's disease. It is well known that there is the influence of smoking on these course of diseases and the number of flares. This influence is different in both diseases and is definitely negative among patients with Crohn's disease. The aim of the study was to establish if there is a difference of smoking influence on the course of disease depending if the patient is the carrier of NOD2/CARD15 mutation or not. 150 patients with CD was examined by careful interview about smoking habits, physically and there was molecular analyze performed of monocytes' DNA. The most often variant of NOD2/CARD15 mutation in Polish population was 802 C>T which causes the conversion of proline into serine in 268 position of protein product. The second most often observed variant was found during the analyze of exon 11 in the temperature of 20 degrees C. There was a frameshift mutation 3020insC present in 14.9% patients with CD which causes the C insertion in 3020 position of protein product. All patients with the frameshift mutation were also carriers of 802C>T mutation. The analysed features were the course of disease, the smoking habit and the difference in group of NOD2/CARD15 carries and patients without the mutation. We took into consideration the fact if the affected person was smoker, ex-smoker or non-smoker. Patients with 802C>T mutation ex-smokers and smokers were older on the onset of the disease (average: 35.8 years), whereas the non-smokers (average: 26.07 years). And what is more interesting, the non-smoking patients were statistically less frequent operated (the partial resection of terminal ileum). Only 31.82% of non-smoking patient with 802C>T mutation were operated.

Transgenic rabbit producing human growth hormone in milk.

The gene construct WAP(6xHisThr):hGH containing the entire human growth hormone gene (hGH) under the rat whey acidic protein (WAP) promoter regulating the expression in mammary glands of mammals was prepared. The 5' end of the gene was modified by the addition of a sequence encoding six histidine residues and a sequence recognized by thrombin. The gene construct was introduced by microinjection into the male pronucleus of a fertilized oocyte. The founder male rabbit was obtained with the transgene mapping to chromosome 7. The presence of the growth hormone was confirmed in samples of milk collected during the lactation of F1 generation females. The growth hormone can be easily purified by affinity chromatography and cleavage by thrombin to an active form.