Growth of C57BL/6 mice and the material and mechanical properties of cortical bone from the tibia.

Murine models are becoming increasingly important for studying skeletal growth and regulation because of the relative ease with which their genomes can be manipulated. This study measured the changes in cortical bone of tibiae from one of the more common models, the C57Bl/6, as a function of aging. A total of 97 mice, male and female, were studied at the ages of 1, 2, 3, 6, 9, and 12 months. The body weight of the animals, the length of the tibiae, the composition (in terms of mineral and organic mass fractions), and the density and modulus of the bone were measured. Peripheral quantitative computed tomography was also used to measure bone mineral density (BMD), total and cortical areas, and the cross-sectional moment of inertia. Most parameters measured followed a growth-like curve, which leveled off some time before 6 months of age. Bone composition and modulus were the same at maturity in both sexes, but there were sex-related differences in the modulus with aging. Dimensional measurements and the density of the bone showed significant differences between male and female animals at all ages, with the male mice having larger values. Skeletal maturity for most factors in C57Bl/6 mice has been reached before the age of 6 months.

Inhibition of bone resorption in vitro and prevention of ovariectomy-induced bone loss in vivo by flurbiprofen nitroxybutylester (HCT1026).

OBJECTIVE: Inhibitors of prostaglandin production, such as nonsteroidal antiinflammatory drugs (NSAIDs), and pharmacologic nitric oxide (NO) donors, such as organic nitrates, have been suggested to protect against bone loss in both humans and experimental animals. Recently, a new class of nitrosylated NSAID (known as NO-NSAIDs) has been developed, which combines the properties of a NO donor with those of a cyclooxygenase (COX) inhibitor. This study investigated the effects of one of these compounds, flurbiprofen nitroxybutylester (HCT1026), on bone metabolism in vitro and in vivo. METHODS: The effects of HCT1026 on osteoclast formation and resorption were determined in vitro using cocultures of primary mouse osteoblasts and osteoclasts. The effect of HCT1026 in vivo was assessed using a mouse model of ovariectomy-induced bone loss. RESULTS: HCT1026 was significantly more efficacious than the parent compound, flurbiprofen, at inhibiting osteoclast formation and bone resorption in vitro, and these effects could not be reproduced by combinations of flurbiprofen with a variety of NO donors. Studies in vivo showed that HCT1026 protected against ovariectomy-induced bone loss by inhibiting osteoclastic bone resorption, whereas flurbiprofen at similar concentrations was ineffective. CONCLUSION: These data indicate that HCT1026 is a potent inhibitor of bone resorption in vitro and protects against ovariectomy-induced bone loss in vivo by a novel mechanism that appears to be distinct from its NO donor properties and from its inhibitory effects on COX activity. We conclude that HCT1026 may be of clinical value in the prevention and treatment of inflammatory diseases such as rheumatoid arthritis, which are characterized by joint inflammation as well as periarticular and systemic bone loss.

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.

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.

Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption.

Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study, we investigated the role that the iNOS pathway plays in mediating the bone-resorbing effects of IL-1 by studying mice with targeted disruption of the iNOS gene. Studies in vitro and in vivo showed that iNOS-deficient mice exhibited profound defects of IL-1-induced osteoclastic bone resorption but responded normally to calciotropic hormones such as 1,25 dihydroxyvitamin D3 and parathyroid hormone. Immunohistochemical studies and electrophoretic mobility shift assays performed on bone marrow cocultures from iNOS-deficient mice showed abnormalities in IL-1-induced nuclear translocation of the p65 component of NFkappaB and in NFkappaB-DNA binding, which were reversed by treatment with the NO donor S-nitroso-acetyl penicillamine. These results show that the iNOS pathway is essential for IL-1-induced bone resorption and suggest that the effects of NO may be mediated by modulating IL-1-induced nuclear activation of NFkappaB in osteoclast precursors.

Estrogen receptor mRNA in mineralized tissues of rainbow trout: calcium mobilization by estrogen.

RT-PCR was undertaken on total RNA extracts from bone and scales of the rainbow trout, Oncorhynchus mykiss. The rainbow trout estrogen receptor (ER)-specific primers used amplified a single product of expected size from each tissue which, using Southern blotting, strongly hybridized with a 32P-labelled rtER probe under stringent conditions. These data provide the first in vivo evidence of ER mRNA in bone and scale tissues of rainbow trout and suggest that the effects of estrogen observed in this study (increased bone mineral and decreased scale mineral contents, respectively) may be mediated directly through ER.

A peptide from the caudal neurosecretory system of the dogfish Scyliorhinus canicula that is structurally related to urotensin I.

Using reversed-phase HPLC in combination with a radioimmunoassay for ovine corticotropin-releasing hormone (CRH), a peptide with CRH-like immunoreactivity was isolated in pure form from an extract of the caudal spinal cord region of the spotted dogfish, Scyliorhinus canicula. The primary structure of the peptide was established as Pro-Ala-Glu-Thr-Pro-Asn-Ser-Leu10-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg- Glu-Met-Ile- Glu20-Ile-Ala-Lys-His-Glu-Asn-Gln-Gln-Met-Gln30-Ala-Asp-Ser- Asn-Arg-Arg-Ile-Met - Asp-Thr40-Ile.NH2. This amino acid sequence shows moderate structural similarity to Catostomus urotensin I (51%) and to human CRH (56%). The data provide, therefore, chemical evidence to support the conclusions of earlier immunohistochemical studies that the diffuse caudal neurosecretory system of elasmobranchs produces a peptide that is immunochemically related to teleost urotensin I peptides. However, the primary structure of urotensin I has been poorly conserved during evolution.

Interleukin-1 beta induces the synthesis of adenylyl cyclase in Swiss 3T3 fibroblasts and MG-63 osteosarcoma cells.

In this study, the longer term (> or = 6 hours) effect of interleukin-1 beta on adenylyl cyclase activity was investigated in Swiss 3T3 and MG-63 cells. No change was evident after 6 hours but after 1, 7 or 15 day incubations a significant increase in basal (1.5- 2 fold) and NaF-stimulated (2-4 fold) adenylyl cyclase activity was observed in interleukin-1 beta pre-treated cell membranes compared with non pre-treated controls. The response to forskolin, a direct stimulus of adenylyl cyclase, was also significantly enhanced, indicating that the effect of interleukin-1 beta was targeted to the enzyme itself. This action of interleukin-1 beta was blocked by co-incubation with cycloheximide, an inhibitor of protein synthesis, which demonstrated that de novo protein synthesis was required. It is concluded that interleukin-1 induces the expression of adenylyl cyclase in 3T3 and MG-63 cells, leading to enhanced activation by NaF and forskolin.

The effect of dietary protein restriction on the secretory dynamics of 1 alpha-hydroxycorticosterone and urea in the dogfish, Scyliorhinus canicula: a possible role for 1 alpha-hydroxycorticosterone in sodium retention.

The putative osmoregulatory role of the unique elasmobranch corticosteroid, 1 alpha-hydroxycorticosterone (1 alpha-OH-B), was investigated using dietary protein restriction as a means of limiting urea biosynthetic ability. Groups of dogfish (Scyliorhinus canicula) were adapted to either a high or a low protein diet (HPD and LPD respectively) and the secretory dynamics of urea and 1 alpha-OH-B were determined following acclimation to normal (100%), 130% and 50% sea water. In normal sea water, LPD fish showed significantly decreased blood production of urea compared with fish fed a HPD (P < 0.05), and the plasma urea concentration required to maintain iso-osmolality was achieved only by a substantial decrease in urea clearance from the plasma. Unlike HPD fish, LPD fish in 130% sea water had no apparent ability to increase plasma urea concentration. An alternative strategy adopted by these animals was the retention of high plasma concentrations of Na+ and Cl-, which increased plasma osmolality and tended to decrease osmotic water loss. Concomitant with the increased ion concentrations, plasma 1 alpha-OH-B concentration was also greatly elevated in LPD fish indicating that the steroid may be acting to minimize Na+ (and Cl-) excretion at osmoregulatory sites such as the rectal gland, kidney and gills. This and a previous study have also demonstrated that 1 alpha-OH-B concentration is elevated in 50% sea water. Decreases in plasma Na+ concentration are tolerated down to 75% sea water, whereafter Na+ is preferentially retained and further decreases in osmolality are achieved by reductions in plasma urea concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of ACTH- and angiotensin II-stimulated 1 alpha-hydroxycorticosterone secretion in the dogfish, Scyliorhinus canicula.

An isolated perifused interrenal gland preparation from the lesser-spotted dogfish, Scyliorhinus canicula, was used to investigate the mechanisms of action of ACTH and angiotensin II (AII) on elasmobranch adrenocortical cells. ACTH-stimulated 1 alpha-hydroxycorticosterone secretion was unaffected by dantrolene and significantly decreased in the absence of extracellular calcium. Dibutyryl cyclic AMP produced a dose-dependent increase in 1 alpha-hydroxycorticosterone secretion. The results suggest that the mechanism of ACTH action in elasmobranchs may be similar to that reported for mammals and amphibians, involving the synergistic action of calcium with the cyclic AMP messenger system. AII-stimulated 1 alpha-hydroxycorticosterone secretion was significantly inhibited in the presence of dantrolene and in the absence of extracellular calcium, indicating that both extracellular and intracellular calcium are required for the full action of AII. These results are consistent with results in mammals and amphibians where AII stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis and changes in intracellular calcium concentration, and they suggest that AII may operate via this mechanism to stimulate 1 alpha-hydroxycorticosterone secretion in elasmobranchs.

Secretory patterns of 1 alpha-hydroxycorticosterone in the isolated perifused interrenal gland of the dogfish, Scyliorhinus canicula.

An isolated in-vitro perifused interrenal gland preparation from the dogfish Scyliorhinus canicula was used to study production of quantitatively the major corticosteroid 1 alpha-hydroxycorticosterone (1 alpha-OH-B), measured by radioimmunoassay. Basal secretory rates were 877.1 +/- 145 (S.E.M.) fmol/mg per 15 min (n = 14) and the preparation remained viable for up to 22 h, as reflected in a brisk response to 10 microM cyclic AMP (cAMP) after this time. Steroid production responded in a dose-dependent manner to porcine ACTH, with 10 microM producing a maximum stimulation of 225% above the basal secretory rate. cAMP (10 microM) produced an increase of 278% above basal, while 1 microM forskolin increased basal secretory rates by 127%. [Val5]- and [Ile5]-angiotensin II (0.1 microM) increased 1 alpha-OH-B production by 120 and 372% respectively over basal secretory rates. Increasing the concentration of K+ in the perfusate from 8 mM to 12, 18, 28 and 40 mM produced a significant rise only at 28 mM. Alterations in the concentration of Na+ and osmolarity of the perifusion medium had inconsistent effects on steroid production. Increased concentrations of urea (from 360 to 720 mM) increased the basal secretory rate by 121%, whilst reducing the concentration of urea (from 360 to 90 mM) had no effect.