J. Cornish

INSULIN INCREASES HISTOMORPHOMETRIC INDICES OF BONE FORMATION IN VIVO

Recent clinical studies have established that bone density is related to both fat mass and circulating insulin levels. A direct action of insulin on the osteoblast may contribute to these relationships. Osteoblast-like cells have insulin receptors, and insulin has been shown to stimulate proliferation of these cells in vitro. However, it has not been possible to study the effects of insulin administration on bone in vivo because of the metabolic effects of insulin, particularly hypoglycemia. A model involving the local injection of insulin over one hemicalvaria of an adult mouse overcomes these difficulties and permits the histomorphometric study of insulins action on bone. Insulin or vehicle was injected daily for 5 days over the right hemicalvariae of adult mice, and the animals were sacrificed 1 week later. All indices of bone formation were significantly increased in imsulin-treated hemicalvariae compared with the noninjected hemicalvariae. There was a 2.73±0.50-fold increase in osteoid area (P=0.0005), a 2.20±0.37-fold increase in osteoblast surface (P=0.021) and a 2.04±0.29-fold increase in osteoblast number (P=0.021). Indices of bone resorption tended to decline and mineralized bone area tended to increase in insulin-treated animals. The direct action of insulin on bone may contribute to the increased bone density seen in obesity and to the osteopenia of type I diabets, conditions associated with insulin excess and deficiency, respectively.

Comparison of the Effects of Calcitonin Gene‐Related Peptide and Amylin on Osteoblasts

Calcitonin gene‐related peptide (CGRP) and amylin are homologous 37‐amino‐acid peptides which have been demonstrated to have anabolic effects on bone. It is not clear whether these effects are mediated by a common receptor, nor is it known which ligand is the more potent. These questions are addressed in the present study using cultures of fetal rat osteoblasts. CGRP increased cell number when present in a concentration ≥10−9 M, but 10−8 M CGRP was required to stimulate thymidine and phenylalanine incorporation. Amylin was effective on these indices at 100‐fold lower concentrations, and its maximal effects were about twice as great as those of CGRP. ED50s for the effects of amylin and CGRP on cell number were 10−12 M and 10−10 M, respectively. There was no additivity between maximal doses of the peptides on these indices. The effects of specific receptor blockers on the maximal stimulation of cell number by these peptides were also studied. The CGRP receptor‐blocker, CGRP‐(8–37), completely blocked the effect of CGRP at blocker concentrations ≥10−9 M. In contrast, the amylin receptor blocker, amylin‐(8–37), completely blocked the effects of CGRP when the blocker was present in concentrations as low as 10−11 M. The KI of CGRP‐(8–37) was 2 × 10−10 M and that of amylin‐(8–37) was 7 × 10−12 M. In converse experiments studying the blockade of maximal doses of amylin, amylin‐(8–37) 10−10 M was effective (KI 1 × 10−10 M), whereas a 100‐fold greater concentration of CGRP‐(8–37) was necessary to achieve the same effect (KI 6 × 10−9 M). It is concluded that amylin and CGRP probably act through a common receptor to stimulate osteoblast growth, and that this receptor has a higher affinity for amylin than for CGRP.

Differential Gene Expression in Cultured Osteoblasts and Bone Marrow Stromal Cells From Patients With Paget's Disease of Bone†

Pagets disease is a focal condition of bone. To study changes in cells within pagetic lesions, we cultured osteoblasts and stromal cells from 22 patients and compared gene expression in these cells to cells from healthy bone. We identified several differentially regulated genes, and we suggest that these changes could lead to the formation of the lesions.

Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes

Peptides purified by HPLC are often in the form of a trifluoroacetate (TFA) salt, because trifluoroacetic acid is used as a solvent in reversed-phase HPLC separation. However, the potential effects of this contaminant in culture systems have not been addressed previously. TFA (10-8 to 10-7 M) reduced cell numbers and thymidine incorporation into fetal rat osteoblast cultures after 24 h. Similar effects were found in cultures of articular chondrocytes and neonatal mouse calvariae, indicating that the effect is not specific to one cell type or to one species of origin. When the activities of the TFA and hydrochloride salts of amylin, amylin-(1-8), and calcitonin were compared in osteoblasts, cell proliferation was consistently less with the TFA salts of these peptides, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding is likely to be relevant to all studies of purified peptides in concentrations above 10-9 M in whatever cell or tissue type. Such peptides should be converted to a hydrochloride or biologically equivalent salt before assessment of their biological effects is undertaken.Peptides purified by HPLC are often in the form of a trifluoroacetate (TFA) salt, because trifluoroacetic acid is used as a solvent in reversed-phase HPLC separation. However, the potential effects of this contaminant in culture systems have not been addressed previously. TFA (10(-8) to 10(-7) M) reduced cell numbers and thymidine incorporation into fetal rat osteoblast cultures after 24 h. Similar effects were found in cultures of articular chondrocytes and neonatal mouse calvariae, indicating that the effect is not specific to one cell type or to one species of origin. When the activities of the TFA and hydrochloride salts of amylin, amylin-(1-8), and calcitonin were compared in osteoblasts, cell proliferation was consistently less with the TFA salts of these peptides, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding is likely to be relevant to all studies of purified peptides in concentrations above 10(-9) M in whatever cell or tissue type. Such peptides should be converted to a hydrochloride or biologically equivalent salt before assessment of their biological effects is undertaken.

Differences in Hip Axis and Femoral Neck Length in Premenopausal Women of Polynesian, Asian and European Origin

There are substantial inter-racial differences in hip fracture incidence. Studies in several different ethnic groups have suggested that differences in the length of the femoral neck may contribute to these. The present study assesses femoral neck and hip axis lengths in three ethnic groups in which it has not been documented previously (Chinese, Indians and Polynesians) and compares these values with those in Europeans. Lengths were measured from dual-energy X-ray absorptiometry scans of the proximal femur in normal premenopausal women (n=225). The Polynesian (1.65 m) and European (1.64 m) women were significantly taller than the two Asian groups (mean height in each, 1.58 m). There were also differences in mean body weight, the Polynesians being the heaviest (76 kg) and the Chinese the lightest (53 kg). Femoral neck lengths were (mean + SD) Chinese 61.5+4.4 mm, Indian 61.5+5.1 mm, Polynesian 68.2+4.3 mm and Europeans 66.0+4.8 mm. Hip axis lengths were Chinese 98.0+5.6 mm, Indian 94.5+5.2 mm, Polynesian 106.4 ± 5.3 mm and European 102.3+5.3 mm. Each of the other groups were significantly different from the Europeans for both variables and, in general, this remained so after height adjustment. These data suggest that shorter femoral necks are common to the major Asian racial groups. However, in contrast to all other ethnic groups studied, Polynesians have longer femoral necks than Europeans and their low incidence of hip fracture is not explicable, therefore, in terms of their femoral neck length. This suggests that either higher bone density or other more subtle differences in proximal femoral geometry must account for the low hip fracture incidence in Polynesians.

Lactoferrin potently inhibits osteoblast apoptosis, via an LRP1-independent pathway.

Lactoferrin induces osteoblast proliferation in vitro and is anabolic to bone in vivo. We recently reported that the low-density lipoprotein-receptor-related protein 1 (LRP1), a multifunctional member of the LDL receptor family, transduces the mitogenic signal activated by lactoferrin. Here we investigate the effects of lactoferrin on osteoblast survival. At periphysiological concentrations (1-10mug/ml), lactoferrin protects both primary rat osteoblastic cells and SaOS2 cells from apoptosis induced by serum withdrawal. Surprisingly, this effect was not sensitive to the LRP1/2 inhibitor receptor-associated protein (RAP). Neither did lactoferrin selectively prevent apoptosis in fibroblastic cells expressing wild-type LRP1 compared to LRP1-null fibroblasts. Lactoferrin activates PI3 kinase-dependent Akt signaling in osteoblasts but this effect is neither LRP1-dependent nor required for lactoferrin-induced cell survival. Lactoferrin activates p42/44 MAPK signaling, but inhibiting this process does not abrogate its pro-survival actions. These results demonstrate that lactoferrin promotes osteoblast survival, an effect that may contribute to its anabolic skeletal actions in vivo. Our data also suggest that the molecular mechanisms that underpin the ability of lactoferrin to promote cell survival differ fundamentally from those which subserve its mitogenic actions, in particular being mediated by a distinct cell-membrane-based receptor.

Lysophosphatidic Acid Is an Osteoblast Mitogen Whose Proliferative Actions Involve Gi Proteins and Protein Kinase C, But Not P42/44 Mitogen-Activated Protein Kinases

The simple glycerophospholipid lysophosphatidic acid (LPA) acts both as an intermediary in phospholipid metabolism and as an intercellular signaling molecule in its own right. In various cell types, LPA signals through its membrane-bound, G protein-coupled receptors to influence cellular processes such as proliferation, survival, and cytoskeletal function. Its actions in bone cells have not been studied. Here we show that the LPA receptor, LPA1/edg-2/vzg-1, is expressed in primary rat osteoblasts and the UMR 106–01 osteoblastic cell line. LPA potently induces DNA synthesis and an increase in cell number in cultures of osteoblastic cells. LPA rapidly (within 10 min) stimulates phosphorylation of p42/44 mitogen-activated protein (MAP) kinases in osteoblastic cells, an effect that is sensitive to inhibition of Gi proteins, inhibition of influx of extracellular calcium, and inhibition of protein kinase C. LPA-induced DNA synthesis is partially inhibited by either pertussis toxin or calphostin C, but is insens...

Failure to Detect Measles Virus Ribonucleic Acid in Bone Cells from Patients with Paget’s Disease

BACKGROUND Pagets disease is a condition of focal accelerated bone turnover. Electron-microscopy investigations of osteoclasts from pagetic lesions have identified nuclear inclusion bodies that have a similar appearance to viral nucleocapsid particles. Subsequently, RNA from several paramyxoviruses has been detected in pagetic tissue, and it was suggested that these viruses, in particular measles, might play a role in the etiology of Pagets disease. We have tested for measles virus sequences in osteoblasts and bone marrow cells collected from pagetic lesions and healthy bone. METHODS Bone and bone marrow samples were taken from Pagets patients and control subjects, and cells were cultured from each of these tissues. RNA was extracted from 13 osteoblast cultures and 13 cultures of bone marrow cells derived from pagetic lesions, and from 26 and 23 control osteoblast and bone marrow cultures, respectively. These samples were sourced from 22 patients with Pagets disease and 31 controls. RT-PCR-nested PCR amplification was used for the detection of the genes for the measles nucleocapsid and matrix proteins. RESULTS Measles virus sequences were not detected in any of the pagetic or control samples. However, measles virus sequences were identified in samples of a measles virus culture isolate included as a positive control, and in a brain sample from a patient with subacute sclerosing panencephalitis, a condition associated with chronic measles infection. CONCLUSION The results of the study do not support the hypothesis that measles virus plays a role in the pathogenesis of Pagets disease.

Dissociation of the effects of amylin on osteoblast proliferation and bone resorption

This study assesses the structure-activity relationships of the actions of amylin on bone. In fetal rat osteoblasts, only intact amylin and amylin-(1-8) stimulated cell proliferation (half-maximal concentrations 2.0 x 10(-11) and 2.4 x 10(-10) M, respectively). Amylin-(8-37), COOH terminally deamidated amylin, reduced amylin, and reduced amylin-(1-8) (reduction results in cleavage of the disulfide bond) were without agonist effect but acted as antagonists to the effects of both amylin and amylin-(1-8). Calcitonin gene-related peptide-(8-37) also antagonized the effects of amylin and amylin-(1-8) on osteoblasts but was substantially less potent in this regard than amylin-(8-37). In contrast, inhibition of bone resorption in neonatal mouse calvariae only occurred with the intact amylin molecule and was not antagonized by any of these peptides. The rate of catabolism of the peptides in calvarial cultures was not accelerated in comparison with that of intact amylin. This dissociation of the actions of amylin suggests that it acts through two separate receptors, one on the osteoclast (possibly the calcitonin receptor) and a second on the osteoblast.This study assesses the structure-activity relationships of the actions of amylin on bone. In fetal rat osteoblasts, only intact amylin and amylin-(1-8) stimulated cell proliferation (half-maximal concentrations 2.0 × 10-11 and 2.4 × 10-10 M, respectively). Amylin-(8-37), COOH terminally deamidated amylin, reduced amylin, and reduced amylin-(1-8) (reduction results in cleavage of the disulfide bond) were without agonist effect but acted as antagonists to the effects of both amylin and amylin-(1-8). Calcitonin gene-related peptide-(8-37) also antagonized the effects of amylin and amylin-(1-8) on osteoblasts but was substantially less potent in this regard than amylin-(8-37). In contrast, inhibition of bone resorption in neonatal mouse calvariae only occurred with the intact amylin molecule and was not antagonized by any of these peptides. The rate of catabolism of the peptides in calvarial cultures was not accelerated in comparison with that of intact amylin. This dissociation of the actions of amylin suggests that it acts through two separate receptors, one on the osteoclast (possibly the calcitonin receptor) and a second on the osteoblast.

Adrenomedullin: a regulator of bone formation

Bone growth and maintenance are highly regulated processes. Throughout life, bone constantly undergoes remodelling, maintaining a balance between bone formation by osteoblasts and bone resorption by osteoclasts. This balance depends on the coordinated activities of many systemic hormones and locally acting factors in the bone microenvironment. Understanding the mechanisms of action of these factors provides a better appreciation of the cellular and molecular basis of bone remodelling. Adrenomedullin has recently been found to stimulate the proliferation of osteoblasts in vitro, and to increase indices of bone formation when administered either locally or systemically in vivo. Adrenomedullin receptors, as well as adrenomedullin itself, are expressed in primary osteoblasts and in osteoblast-like cell lines. In this paper we discuss the anabolic effect of adrenomedullin in bone, and present new evidence for a possible role of adrenomedullin in the regulation of cartilage cells. We show that adrenomedullin stimulates proliferation of primary chondrocytes in culture and that mRNA for adrenomedullin and for adrenomedullin receptors are expressed in these cells. Studies of structure-activity relationships have demonstrated that osteotropic effects of adrenomedullin can be retained in peptide fragments of the molecule which lack the parent molecules vasodilatory properties. Thus, these small peptides, or their analogues, are attractive candidates as anabolic therapies for osteoporosis.