An assessment of the anabolic skeletal actions of the common-region peptides derived from the CGRP and calcitonin prohormones.
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Animals↗
Biomedical subjects
G A Howard
Publications and source records attributed to G A Howard.
17 beta estradiol stimulation of endosteal bone formation in the ovariectomized mouse: an animal model for the evaluation of bone-targeted estrogens.
To evaluate the potential of an animal model as a means to identify bone-targeted estrogens, we have studied the response of the skeleton of ovariectomized mice to prolonged estrogen treatment. Seventy female Swiss-Webster mice were randomly divided into ten groups, with nine groups undergoing bilateral ovariectomy and one group a sham procedure. Mice were injected subcutaneously once per week for nine weeks with one of the following doses of estrogen (17 beta-E2) in oil vehicle: 19.3, 38.5, 75, 150, 300, 500, 1000, or 3000 micrograms. One group of ovariectomized (OVX) mice and the sham operated animals received vehicle injections only. At the end of the nine-week experimental protocol, there were no significant differences in body weights among any treatment groups. However, when compared to control values, spleen weights in vehicle-treated OVX mice and in mice treated with 1000 micrograms or 3000 micrograms of 17 beta-E2 were significantly elevated (p less than .01). Liver weights in the OVX mice treated with 1000 or 3000 micrograms 17 beta-E2 were also increased significantly (p less than .05). Comparisons of uterine weights and cortical bone areas were strongly correlated with 17 beta-E2 dose (r2 = .86 and .94, respectively), with maximal increases observed at estradiol doses in excess of 500 micrograms per week. Furthermore, based on bone histomorphometry of in vivo fluorochrome labels, increases in cortical bone area could be attributed to accelerated rates of endosteal mineral apposition and bone formation. These results indicate that the comparison of the response of endosteal bone and uterine tissue in the OVX mice to chronic estrogen treatment offers the potential to identify estrogen and/or estrogen-like compounds with bone-specific activity.
Animals↗
Hormonal regulation of intercellular communication: parathyroid hormone increases connexin 43 gene expression and gap-junctional communication in osteoblastic cells.
The presence of gap junctions between osteoblastic cells has been previously reported. For this study we used the rat osteosarcoma cell line UMR 106, which expresses the osteoblastic phenotype, as a model to characterize further the nature, physiology, and regulation of gap junctions. Northern blot analysis identified a 3.0-kilobase RNA species corresponding to the gap junction protein connexin 43. The presence of two other connexin RNA species (26 and 32) could not be detected by this method in these cells. The identified connexin RNA was amplified by reverse transcription coupled to polymerase chain reaction; the sequence of the amplified product appears identical to the sequence of a cloned rat heart connexin 43 gene. After treatment with PTH, forskolin, and 8-Br-cAMP (a cAMP analog), the levels of connexin 43 RNA in UMR 106 cells increased. Further evidence for the role of PTH and cAMP in the physiology of gap junctions in these cells was obtained with Lucifer yellow dye transfer experiments. Gap-junctional intercellular communication increased in response to PTH and forskolin (an inducer of adenylate cyclase activity). Expression of connexin 43 RNA increased severalfold in response to PTH in a concentration- and time-dependent fashion. Connexin 43 RNA and its PTH-mediated stimulation were also observed in several other osteoblastic cell lines. The roles of PTH and forskolin in regulating the physiological state of gap junctions were confirmed in primary cultures of rat calvaria osteoblasts.
8-Bromo Cyclic Adenosine Monophosphate↗
Bone-cell changes in estrogen-induced bone-mass increase in mice: dissociation of osteoclasts from bone surfaces.
Early studies in certain avian and mammalian species have described estrogen-associated bone-cell changes, which were based on bone cells that were neither quantified nor identified histochemically (osteoclasts). In the experiments described here, weanling female mice were given a pharmacological dose of 17 beta-estradiol benzoate (1 mg/week) for 1 and 4 weeks, and changes in osteoclasts and osteoblasts were assessed in tibial metaphyses and diaphyses. In the proximal metaphysis, the number of osteoclasts/mm surface length was significantly reduced by estrogen at 1 week (43%) and 4 weeks (64%), which was accompanied by significant increases in the number of osteoclasts in the marrow space not in contact with bone surfaces (no./mm2: 382% and 999%, respectively). These increases, along with observations of decreased osteoclast size (19%), of changes in osteoclast morphology, and of numerous acid-phosphatase-positive fragments in the marrow space, suggest that estrogen treatment causes the dissociation and disintegration, and thus decreased activity, of osteoclasts. The above changes were accompanied by more than 48% increases in the number of trabecular osteoblasts. In the diaphysis, the number of endosteal osteoclasts was significantly decreased by estrogen at 1 week (32%), but was not significantly changed at 4 weeks. These changes were attended by significant increases in the number of osteoclasts in the marrow space not in contact with bone surfaces (no./mm2: 393% at 1 week and 342% at 4 weeks). The number of endosteal osteoblasts was also increased by estrogen at 1 week (132%), and so was the size of endosteal osteoblasts (39% at 1 week and 81% at 4 weeks). Comparable results were obtained when a lower dose of 17 beta-estradiol benzoate (20 micrograms/week) was given to ovariectomized mice. The increase in bone mass and its associated cell changes following estrogen treatment were also found in athymic nude mice, suggesting that these bone/bone cell changes are independent of the thymus.
Animals↗
Ethanol inhibits human bone cell proliferation and function in vitro.
The direct effects of ethanol on human bone cell proliferation and function were studied in vitro. Normal human osteoblasts from trabecular bone chips were prepared by collagenase digestion. Exposure of these osteoblasts to ethanol in concentrations of 0.05% to 1% for 22 hours induced a dose-dependent reduction in bone cell DNA synthesis as assessed by incorporation of 3H-thymidine. After 72 hours of ethanol exposure in concentrations of 0.01% to 1%, protein synthesis as measured by 3H-proline incorporation into trichbroacetic acid (TCA)-precipitable material was reduced in a dose-dependent manner. Human bone cell protein concentrations and alkaline phosphatase total activity were significantly reduced after exposure to 1% ethanol for 72 hours, but not with lower concentrations of ethanol. This reduction in osteoblast proliferation and activity may partially explain the development of osteopenia in humans consuming excessive amounts of ethanol.
Alkaline Phosphatase↗
In vivo actions of insulin-like growth factor-I (IGF-I) on bone formation and resorption in rats.
The in vivo action of insulin-like growth factor-I on bone metabolism has been studied using a new model. Insulin-like growth factor-I (IGF-I) was continuously infused into the arterial supply of the right hindlimb of ambulatory rats for up to 14 days and the effects on cortical and trabecular bone formation and the number of osteoclasts were determined by histomorphometric techniques. The contralateral limb acted as an internal control. IGF-I infusion significantly increased cortical bone formation (p less than 0.01). Trabecular bone was increased 22% (p = 0.07), but the infusion was only for seven days. These effects of IGF-I were age dependent, being absent in young, rapidly growing animals, but present at least until one year of age. IGF-I appears to be a purely anabolic hormone for bone formation, since it significantly stimulates osteoblasts and decreases the number of osteoclasts. Thus, although IGF-I mediates the growth-promoting effect of growth hormone, it does not mediate growth hormone's action on bone resorption.
Animals↗
Identification of a low molecular weight inhibitor of osteoblast mitogenesis in uremic plasma.
A low molecular weight inhibitor of cartilage sulfation has been detected in the plasma of dialysis patients. Preliminary studies of this inhibitor have suggested that it may have a role in decreasing bone mass, possibly by suppressing bone cell proliferation. Since the in vitro bioassay of crude sulfation inhibitor preparations is relatively nonspecific, we investigated whether there might also be an inhibitor of osteoblast mitogenesis in uremic plasma. We fractionated plasma and plasma ultrafiltrates from dialysis patients by gel filtration chromatography and looked for inhibition of mitogenesis in cultured osteoblasts. Material from fractions with a molecular weight range of 750 to 900 inhibited osteoblast mitogenesis. The inhibitory effect, however, could be overcome with serum or insulin-like growth factor-I, suggesting that the mechanism of inhibition was not growth factor dependent. Further characterization of the inhibitor revealed that it was not a peptide or a polar lipid. We conclude that uremic plasma contains a bone cell mitogenic inhibitor which may have a role in regulating bone remodeling in adults and bone growth in children.
Cell Division↗
Acetate inhibition of chick bone cell proliferation and bone growth in vitro.
A hypothesis has been advanced that parenteral solutions as commonly formulated for use in clinical practice have a toxic effect on cell metabolism. A specific component of these solutions, sodium acetate, has been suggested to disrupt normal bone turnover and therefore to contribute to the osteopenia observed in patients receiving hemodialysis and parenteral nutrition (PN). We developed an in vitro model to test the hypothesis that sodium acetate at concentrations that are infused in PN solutions has a deleterious effect on bone metabolism. Osteoblasts and preosteoblasts from 16- to 17-day-old embryonic chick calvaria, and tibiae and femora from 10-day-old embryonic chicks were grown in BGJb medium (control) or in BGJb medium plus sodium acetate (5, 10, or 20 mM). Calvarial cell proliferation was quantified by direct cell counts as well as by incorporation of [3H]TdR into DNA as an index of cell proliferation. Calvarial cell alkaline phosphatase activity was quantified by the ability of extracts of the cultured cells to hydrolyze p-nitrophenyl phosphate to p-nitrophenol, and bone growth was determined by measuring final dry weight. Calvarial cell counts as well as DNA synthesis showed a dose-dependent decrease in the presence of sodium acetate (5-20 mM) compared with controls. [3H]TdR incorporation was decreased a mean 19% with 5 mM, 38% with 10 mM, and 63% with 20 mM acetate. Alkaline phosphatase activity per cell increased 48% with 5 mM, 140% with 10 mM, and 355% with 20 mM acetate. Cell viability as assessed by trypan blue exclusion was identical for test and control media (greater than 95%).(ABSTRACT TRUNCATED AT 250 WORDS)
Acetates↗
Prostaglandins in inflammatory bone pathology: mechanism and therapeutic benefit of etodolac.
To investigate the role of PGE2 in the development of bone and joint pathology in rat adjuvant arthritis, hindlimb paws were evaluated by calcified tissue histologic techniques focusing on histochemical visualization of cartilage and bone lesions. Case studies of hindlimbs from normal, adjuvant arthritic, and etodolac-treated arthritic rats demonstrated the association of disease severity with inflammation, chondromalacia, replacement of adipose bone marrow with a fibroid marrow, osteoclastic bone resorption, synovial cysts, and pannus formation within the joints. Extensive periosteal intramembranous bone formation was temporally associated with joint destruction and medullary tissue pathology. In vivo data were correlated with in vitro effects of inflammatory mediators (IL-1, PGE2) on bone resorption. Etodolac blocked bone explant PGE2 accumulation at concentrations of 10(-7) M and higher, and inhibited bone resorption at concentrations of 10(-5) M and higher. The data indicate that in vitro and in vivo models of bone metabolism are well correlated regarding prostaglandin synthesis; that the inflammatory mediator PGE2 is largely responsible for the involvement of skeletal tissue in the adjuvant arthritis model; and that the effects of etodolac are specifically mediated by its ability to inhibit PGE2 accumulation in vivo.
Animals↗
Procalcitonin's amino-terminal cleavage peptide is a bone-cell mitogen.
The parafollicular-cell (C-cell) hormone calcitonin (CT) can preserve or even augment skeletal mass by inhibiting osteoclast-mediated bone resorption. The possibility of an additional anabolic skeletal influence has also been raised: C cells might, via CT or other secretory products, affect osteoblast-mediated bone formation. The 57-residue amino-terminal procalcitonin cleavage peptide, N-proCT, has recently been identified in human and rat C cells, where it is made and secreted in equimolar amounts with CT. The coelaboration of N-proCT and CT and N-proCT's sequence conservation during evolution prompted us to investigate the potential skeletal bioactivity of N-proCT. We found that synthetic human N-proCT, at nanomolar concentrations, stimulated proliferation of normal and neoplastic human osteoblasts. At maximally effective doses, human N-proCT caused more than a 100% increase above the control rate of DNA synthesis, an effect comparable to the maximal growth effect of insulin, a potent mitogen for osteoblasts. Human N-proCT exerted a similar maximal mitogenic effect in chicken osteoblast cultures but at 1000-fold greater concentrations than in human bone-cell cultures. The bone-cell action of N-proCT was potentiated with insulin with a greater than 200% increase in DNA synthesis at high insulin concentrations. In sharp contrast to these findings for N-proCT, the other bioactive C-cell peptides, CT and somatostatin, showed no mitogenic effects in human or chicken osteoblast cultures. Our results indicate that the action of N-proCT on cultured bone cells is separate from and potentiated by insulin, a known growth factor. Unlike insulin and related growth factors such as insulin-like growth factor I, N-proCT is not mitogenic in skin fibroblast cultures. We propose that N-proCT is a C-cell hormone that promotes bone formation via stimulatory actions on osteoblasts and preosteoblasts.
Amino Acid Sequence↗
Ontogeny of peptidylglycine alpha-amidating monooxygenase activity in rapidly mineralizing bone from neonatal mouse.
Peptidylglycine alpha-amidating monooxygenase is hormonally, developmentally, and nutritionally regulated. In several tissues concomitant changes in enzyme activity and the level of expression of a known amidated peptide have been demonstrated. We report that neonatal mouse calvarium, a rapidly mineralizing bone, has detectable amidation enzyme activity. The level of activity varied 3-10-fold during the first 9 d of life. Production of one or more amidated peptides by bone may be coordinately regulated.
Aging↗
Parathyroid hormone potentiates the effect of insulin-like growth factor-I on bone formation.
Insulin-like growth factor-I and parathyroid hormone are both known regulators of bone formation. In this study, human recombinant IGF-I and bovine PTH (1-34) and their combination were studied for their effects in vitro on the proliferation of embryonic chick osteoblast-like cells (osteoblasts) and in vivo on bone formation in normal rats. Osteoblasts from 17-day-old chick embryos were cultured in serum-free BGJb medium containing 0.1% bovine albumin. After 2 days, IGF-I and/or PTH were added. Twenty-four hours later [3H]thymidine incorporation into trichloroacetic acid precipitable material was quantified as an index of cell proliferation. This has previously been shown to reflect actual cell division. IGF-I at doses ranging from 0.85 to 13.6 nmol/l caused a dose-dependent increase in [3H]thymidine incorporation into osteoblasts. PTH alone (10 to 1000 pmol/l) had no significant effect. However, when combined with IGF-I, PTH potentiated the mitogenic effect of IGF-I and achieved statistical significance at 30 and 100 pmol/l (p less than 0.05). This potentiation was also studied in vivo. The right hind-limbs of rats weighing 150 g were infused intra-arterially by an osmotic minipump with graded doses of IGF-I (0.1 to 0.4 nmol/day) and/or PTH (0.27 nmol/day) for 7 days. The rate of trabecular bone apposition (formation) was measured by double tetracycline labelling and compared with the contralateral uninfused limb which acted as the control. Histomorphometric data revealed that neither IGF-I nor PTH alone had a significant effect on trabecular bone apposition rate compared with control limbs.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
In vivo calvarial bone cell responses to dietary perturbations and the implications for mineral homeostasis.
Calvariae from small animals have been an important source for in vitro studies of bone. However, few in vivo studies have been undertaken on quantitative cell changes in calvariae. In the present study of mineral perturbations, rats were first deprived of calcium. After 18 days endosteal osteoclasts and nuclei/osteoclast in the parietal bone had increased 120% (P less than 0.001) and 26% (P less than 0.001), respectively, the marrow space had increased 141% (P less than 0.001), and the bone area experienced a 49% decrease (P less than 0.001). This thinning and weakening of the calvaria was accompanied by a compensatory increase in the number of endosteal osteoblasts (297%, P less than 0.001). These rats were then replenished with calcium, and after 14 days the number of endosteal osteoclasts had decreased to 86% (P less than 0.001) below the control and the endosteal surface was almost completely covered by osteoblasts (866% above the control, P less than 0.001). Bone area was increased by 51% (P less than 0.01). Similarly, in calcium-deficient rats in the tibial diaphysis at the fibular junction, the number of endosteal osteoclasts and the medullary space increased 1606% (P less than 0.001) and 63% (P less than 0.001), respectively, which were accompanied by a 32% decrease (P less than 0.001) in cortical bone area. After calcium replenishment, most endosteal osteoclasts in the tibial diaphysis disappeared from the endosteal surface and were replaced by osteoblasts (increased 487%, P less than 0.001). These results indicate that changes in bone cell activity in response to calcium deficiency are similar in calvariae and long bones, and that mobilization of calcium from the calvaria during calcium deficiency occurs at the expense of the protective action of the calvaria. Therefore, long bones as well as membranous bones are apparently important for the maintenance of mineral homeostasis.
Animals↗
Production of granulocyte-stimulating and bone cell-modulating activities from a neutrophilia hypercalcemia-inducing murine mammary cancer cell line.
We have previously shown that a transplantable murine tumor (CE mammary carcinoma) causes marked neutrophilia and excessive bone resorption in vivo. In order to understand the humoral mechanism of these tumor-induced phenomena, we successfully established a cell line (CE 816) and subsequently cloned CE mammary carcinoma cells in serum-free culture conditions. Cultured CE tumor cells continued to induce neutrophilia and hypercalcemia when they were transplanted back into mice. Conditioned medium (CM) prepared from the CE 816 cell line and control non-neutrophilia-inducing tumor cells were evaluated for stimulation of neutrophilic colony formation, embryonic bone cell proliferation, and bone resorption in vitro assays. Both the CE 816 and control tumor CM demonstrated colony-stimulating activity, but the CE 816 CM stimulated more neutrophilic colonies than the control tumor at all experimental conditions examined. The CE 816 CM demonstrated bone-resorbing activity but not control tumor CM. Both types of CM stimulated proliferation of embryonic bone cells. Production of colony-stimulating and bone-mitogenic activities was directly related to the tumor cell growth in vitro. CM prepared from four clones of CE tumor cells demonstrated both colony-stimulating and bone cell-mitogenic activities. These studies provided evidence that CE mammary carcinoma cells produce factors affecting granulopoiesis and bone cells in vitro, and these activities are clonal in origin.
Animals↗
Synthesis of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by calvarial cells. Characterization of the enzyme systems.
The synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] from 25-hydroxycholecalciferol [25(OH)D3] has previously been shown to occur in cells isolated from bone. The main findings of the present study are that the enzyme systems which catalyse these syntheses are: (1) active at 'in vitro' substrate concentrations over the range of 2-50 nM; (2) regulatable in a complex way by 1,25(OH)2D3, 24,25(OH)2D3, 25,26-dihydroxycholecalciferol and 25(OH)D3, but not by cholecalciferol ('vitamin D3'); and (3) have relatively short half-lives (approx. 5 h).
24,25-Dihydroxyvitamin D 3↗
Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities.
Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)
Acid Phosphatase↗
Reactivation of inhibited bone acid phosphatase and its significance in bone histomorphometry.
Despite biochemical demonstration of acid phosphatase (AcP) activation or reactivation in bone, few attempts have been made to show similar effects histochemically. Bones from growing rats, when fixed in 4% buffered formaldehyde at room temperature and demineralized in 5% formic acid, exhibited expected inactivation of AcP. The inhibited AcP, however, was reactivated by pre-incubation of sections for 1 hr at 37 degrees C in the following buffers: 0.2 M Tris, 0.2 M glycine, 0.2 M NaHCO3, or 0.1 M borax, as well as in alkaline water, but not in 0.2 M Na2HPO4 (all at pH 9). The reactivation was (a) site-specific (e.g., osteoclasts, osteoblasts, osteocytes, and cement lines), (b) temperature- and pH-dependent, (c) unaffected by OH- or SH--binding agents or by an alkaline phosphatase inhibitor, and (d) inhibited completely by 10 mM Na2HPO4. The reactivation process, much simplified and/or more effective than with the methods previously reported, was observed in all 83 human biopsy bones embedded in methyl methacrylate and in human bones stored in cold buffered formaldehyde for 7 months. This study demonstrates a unique method for reactivating and thus localizing the inhibited AcP in bones, and suggests possible applications in bone histomorphometry.
Acid Phosphatase↗
Development and reversibility of aluminum-induced bone lesion in the rat.
We studied the effect of aluminum injections on bones of rats after intervals of 3, 6, and 9 weeks. To study reversibility, we allowed one group to recover for 3 weeks. Both weanling and adult rats were examined to determine the influence of age. The calcium, phosphate, creatinine, and parathyroid hormone levels were similar in aluminum-treated rats and controls. Aluminum could be seen by histochemical stain after 6 weeks, but at that time the bone was otherwise normal. By 9 weeks the bone formation (as measured by tetracycline labeling) in aluminum-treated rats was severely decreased on trabecular and endosteal surfaces. The periosteal surfaces showed normal formation. After 3 weeks of recovery, the bone formation rate in the young aluminum-treated rats was similar to that in the controls, although the serum and bone aluminum values had not significantly decreased. A higher percentage of aluminum was seen in the cement lines. In the adult rats, the bones had more stainable aluminum, and increased osteoid was noted along trabecular and periosteal surfaces. The doses of aluminum used in these rats greatly exceeded those that cause toxicity in humans; thus these findings may not directly apply to clinical practice. We conclude that aluminum administration can lead to decreased rates of bone formation in the rat, despite normal calcium level and renal function, and without decreased parathyroid hormone levels. The peritoneal route of administration could also have contributed to bone lesions by causing peritonitis, malabsorption, or both. Adult rats showed signs of early osteomalacia.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗