Operational Head-on Beam-Beam Compensation with Electron Lenses in the Relativistic Heavy Ion Collider.

Head-on beam-beam compensation has been implemented in the Relativistic Heavy Ion Collider in order to increase the luminosity delivered to the experiments. We discuss the principle of combining a lattice for resonance driving term compensation and an electron lens for tune spread compensation. We describe the electron lens technology and its operational use. To date, the implemented compensation scheme approximately doubled the peak and average luminosities.

[What Makes Happy Doctors? Job Satisfaction of General Practitioners in Mecklenburg-Western Pomerania - a Representative Cross-sectional Study]. / Was macht Ärzte glücklich? Berufszufriedenheit von Hausärzten in Mecklenburg-Vorpommern - eine repräsentative Querschnittsstudie.

AIM: Studies provide evidence for the importance of general practitioners (GPs) job satisfaction for a secure and high quality health care provision. This study focuses on job satisfaction of GPs in Mecklenburg-Western Pomerania (MV), a rural area threatened by a lack of GPs. We investigate how satisfied GPs are with their job and which factors influence their job satisfaction. METHODS: All 1 133 GPs working in MV in December 2011 were asked to complete a 57-item-questionnaire. The response rate reached 50.1%. RESULTS: The sample is representative for GPs in MV. Levels of job satisfaction are high and correlate with age and sex: females and GPs below 50 years of age are more satisfied. Factors contributing to high job satisfaction include a good doctor-patient relationship, fair pay, and the variety of reasons for doctor-patient consultations in primary care. Although all GPs were dissatisfied with bureaucracy, this factor has little impact on GPs' overall job satisfaction. CONCLUSION: In light of the imminent lack of GPs, in future it will be important to improve factors that have been demonstrated to increase job satisfaction.

A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide.

The complementary DNA encoding a 585-amino acid parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor with seven potential membrane-spanning domains was cloned by COS-7 expression using an opossum kidney cell complementary DNA (cDNA) library. The expressed receptor binds PTH and PTHrP with equal affinity, and both ligands equivalently stimulate adenylate cyclase. Striking homology with the calcitonin receptor and lack of homology with other G protein-linked receptors indicate that receptors for these calcium-regulating hormones are related and represent a new family.

DNA synthesis is not necessary for osteoclastic responses to parathyroid hormone in cultured fetal rat long bones.

Osteoclasts, the principal cells mediating bone resorption, are believed to increase their size, number, and resorbing activity in response to parathyroid hormone (PTH) through mechanisms dependent upon the fusion of specific mononuclear precursor cells into either new or existing multinucleated osteoclasts. To address the question of whether these actions of PTH are dependent on the replication of osteoclast precursor cells, we examined the ability of an inhibitor of DNA synthesis, hydroxyurea (HU), to alter bone resorption, osteoclast formation, and DNA synthesis in cultured fetal rat bones treated with PTH. We found that HU significantly reduced [3H]thymidine incorporation into the bones and labeling of osteoclast nuclei by greater than 90%, but did not prevent PTH from stimulating bone resorption, measured as the release of 45Ca, or from increasing the number of osteoclasts in the bones. In bones cultured without PTH, HU decreased the rate of bone resorption, but not the number of osteoclasts per bone. We conclude that in fetal rat bone cultures, PTH can increase osteoclast number and stimulate bone resorption by affecting existing osteoclasts and osteoclast precursors, and that replication of osteoclast precursor cells is not necessary for PTH to stimulate a resorptive response. In unstimulated cultures it appears that HU inhibits bone resorption by affecting mechanisms that are independent of changes in osteoclast number and that may be influenced by cell replication or other unknown factors.

Colony-stimulating factors regulate the development of multinucleated osteoclasts from recently replicated cells in vitro.

Osteoclasts mediate the process of bone resorption. However, little is known about the mechanisms that regulate the formation of either osteoclasts or osteoclast precursors. In contrast, colony-stimulating factors (CSFs) are well-known to regulate the formation of myeloid cells and their precursors. Because osteoclasts and myeloid cells may originate from a common stem cell, we examined the effects of two CSFs, granulocyte-macrophage CSF (GM-CSF) and interleukin 3 (IL-3), on bone resorption, osteoclast formation, and the incorporation of recently replicated nuclei into the osteoclasts of mouse bone cultures. CSFs had little effect on the formation rate of osteoclasts or their resorptive activity but significantly decreased the percentage of recently replicated osteoclast progenitor cell nuclei present in the osteoclasts of bones treated with parathyroid hormone. GM-CSF also increased the number of myeloid cells in the marrow space of the cultures and the percentage of these cells derived from recently replicated progenitors. These results demonstrate that GM-CSF and IL-3 can regulate the development of osteoclasts from recently replicated precursor cells in cultured fetal mouse long bones. However, the mechanisms by which CSFs influence osteoclast formation are difficult to determine from these studies because markers for the osteoclast progenitor and precursor do not exist. These data also provide evidence that the differentiation of osteoclast progenitors is regulated by different factors at different points in their ontogeny.

Neurofibromin and its inactivation of Ras are prerequisites for osteoblast functioning.

Skeletal problems and osteoporosis occur in up to 50% affected neurofibromatosis type 1 (NF1) humans. Inactivation of neurofibromin results in deregulation of Ras signal transduction. Little is known of bone biology in humans with NF1. The goal of our work was to determine if loss-of-function of Nf1 gene was associated with altered bone homeostasis and Ras signal transduction. Because homozygous Nf1 mice are embryonically lethal, heterozygote Nf1 (Nf1+/-) male mice were used to investigate skeletal phenotypes and osteoprogenitor functions, using standard in vivo and in vitro assays. We found that bone mass and geometry of Nf1+/- mice did not differ from wild type controls, despite a trend to less bone formation. Nf1+/- committed osteoprogenitors from femur metaphysis exhibited premature apoptosis and higher proliferation. Ras signaling was activated in primary Nf1+/- bone marrow-inducible osteoprogenitors. Inducible osteoprogenitors exhibited lower induction of osteoblast differentiation, assessed as alkaline phosphatase positive CFU-f. A screen of osteoblast marker genes showed a selective increase in osteopontin (OPN) mRNA and protein expression in these cells. OPN protein was increased in Nf1+/- bone, especially in cortical bone matrix. Because bone cell abnormalities in Nf1 haploinsufficiency were detected in vitro, redundant pathways must compensate for the deregulation of Ras signaling in vivo to maintain normal bone mass and function in vivo. Our in vitro data revealed that neurofibromin and its control of Ras signaling are required for osteoprogenitor homeostasis.

Identification of osteocalcin mRNA in nonosteoid tissue of rats and humans by reverse transcription-polymerase chain reaction.

Diseased or necrotic tissue can become calcified in a way that resembles bone. We examined soft tissues for the presence and regulation of the mRNA for the bone-associated protein, osteocalcin (OC). RNA was isolated from liver, kidney, lung, brain, muscle, and bone of young (2 months) male SD rats and analyzed for beta-actin, IGF-I, metallothionein IIa, alpha 1 collagen, calbindin-D9k (CaBP), and OC mRNA by reverse transcription-polymerase chain reaction (RT-PCR). All PCR products but CaBP were found in bone; CaBP was present only in duodenum, kidney, and lung. OC product was detected in all tissues; the identity of the PCR product was confirmed by sequencing. Bone OC mRNA levels were calculated to be 1000-fold higher than duodenal levels. Rats fed a 0.8% strontium diet for 7 days to drive down serum 1,25-dihydroxyvitamin D3 levels [1,25(OH)2D3] and then injected with 300 ng 1,25(OH)2D3/100 body weight had increased duodenal CaBP (2.5-fold) and femur OC mRNA (2.2-fold) 24 h after treatment. Duodenal OC mRNA was unchanged. OC mRNA was found in nondiseased human aortae, and the amount of message was elevated in calcified aorta and calcified aortic plaques. These results demonstrate that (1) tissues other than bone have low basal expression of OC mRNA, (2) OC mRNA is not regulated by vitamin D in nonosteoid tissue, and (3) expression of OC mRNA in atherosclerotic aorta reflects a role for bone-forming cells in ectopic bone formation observed in certain disease conditions.

Effects of continuous and intermittent administration and inhibition of resorption on the anabolic response of bone to parathyroid hormone.

The role of resorption in the anabolic response of bone to parathyroid hormone (PTH) is not well understood. In contrast to the increase in bone mass induced by intermittent PTH in intact rats, continuous infusion of PTH into thyroparathyroidectomized (TPTX) rats failed to increase bone volume. The objective of this study were to determine if continuous infusions of low doses of PTH were anabolic in intact rats and if inhibition of resorption would enhance or block an anabolic action of PTH. Young male rats were treated with either continuous infusion or intermittent injections of hPTH-(1-34) for 12 days. In experiment 1, PTH, infused daily at 4 micrograms per 100 g, increased femur calcium and dry weight. Unlike infusion of 8 micrograms PTH, which did not alter bone mass, intermittent PTH at 8 micrograms was anabolic and increased bone mass by increasing trabecular thickness and number. Infusion of 16 micrograms induced hypercalcemia and death. In experiment 2, lower dose daily infusions of 0.25-4 micrograms PTH per 100 g did not increase bone mass. In experiment 3, in rats pretreated with dichloromethylene diphosphonate (Cl2MDP) to inhibit resorption and subsequently exhibiting decreased bone formation, PTH, irrespective of the method of administration, reversed the inhibitory effects of Cl2MDP on bone formation. Thus, intermittent and continuous PTH increase bone formation independently of effects on bone resorption, but only intermittent PTH increases bone mass consistently.

Nuclear matrix proteins and osteoblast gene expression.

The molecular mechanisms that couple osteoblast structure and gene expression are emerging from recent studies on the bone extracellular matrix, integrins, the cytoskeleton, and the nucleoskeleton (nuclear matrix). These proteins form a dynamic structural network, the tissue matrix, that physically links the genes with the substructure of the cell and its substrate. The molecular analog of cell structure is the geometry of the promoter. The degree of supercoiling and bending of promoter DNA can regulate transcriptional activity. Nuclear matrix proteins may render a change in cytoskeletal organization into a bend or twist in the promoter of target genes. We review the role of nuclear matrix proteins in the regulation of gene expression with special emphasis on osseous tissue. Nuclear matrix proteins bind to the osteocalcin and type I collagen promoters in osteoblasts. One such protein is Cbfa1, a recently described transcriptional activator of osteoblast differentiation. Although their mechanisms of action are unknown, some nuclear matrix proteins may act as "architectural" transcription factors, regulating gene expression by bending the promoter and altering the interactions between other trans-acting proteins. The osteoblast nuclear matrix is comprised of cell- and phenotype-specific proteins including proteins common to all cells. Nuclear matrix proteins specific to the osteoblast developmental stage and proteins that distinguish osteosarcoma from the osteoblast have been identified. Recent studies indicating that nuclear matrix proteins mediate bone cell response to parathyroid hormone and vitamin D are discussed.

Osteoprogenitor cells as targets for ex vivo gene transfer.

We transduced osteoprogenitor cells with recombinant retrovirus and analyzed proviral integration patterns into chromosomal DNA to detect for the first time the clonal and cellular fate of osteoprogenitor-derived progeny cells. Metaphyseal bone cells and diaphyseal stromal cells were isolated from the distal femurs of young rats, transduced with the vM5neolacZ recombinant retrovirus, and selected in the neomycin analog, G418. Following surgical marrow ablation of a femur in one leg of mature rats, retroviral-transduced metaphyseal or diaphyseal cells were injected into the ablated site. These rats were killed 5-6 days later. Metaphyseal and diaphyseal cells were isolated from distal femurs, selected in G418, and stained for beta-galactosidase (beta-gal+). The number and clonal origin of transduced progenitor cells were determined. High numbers of beta-galactosidase colonies with an osteoblast phenotype were obtained following metaphyseal transplants and detected in 100% of metaphyseal and none of diaphyseal specimens. In contrast, beta-galactosidase colonies derived from diaphyseal transplants were detected in 50% of specimens in both the metaphysis and diaphysis, and the absolute number of progenitor cell colonies was 60-fold less than metaphyseal transplants. Provirus was only detected in the ablated bones and not in the contralateral bone or other tissues. Proviral integration fragment analysis showed a single integration site for recovered metaphyseal cell clones, consistent with their origination from a common single progenitor. This is one of the first demonstrations of successful transplantation of clonal osteoprogenitors to their site of origin in bone. It may be possible to use these cells to target genes to bone for therapeutic use in skeletal and hematopoietic diseases.

Resorption is not essential for the stimulation of bone growth by hPTH-(1-34) in rats in vivo.

Chronic low doses of hPTH-(1-34) stimulate bone growth in rats in vivo. The objective of these studies was to determine if the anabolic effect of hPTH-(1-34) on rat bone in vivo is dependent on an initial stimulation of resorption by blocking resorption with either salmon calcitonin (CT) or dichloromethylene diphosphonate (Cl2MDP). Male Sprague-Dawley rats, 70-100 g, were treated with daily subcutaneous (SC) injections of vehicle (V) or hPTH-(1-34), 8 micrograms per 100 g (PTH), for 12 days. In experiment 1, rats were given CT for 3 (CT3) or 12 (CT12) days, either alone or in combination with hPTH-(1-34) (CT3-PTH and CT12-PTH) or vehicle for 12 days. In experiment 2, rats were pretreated for 4 days with Cl2MDP or its vehicle before starting the daily PTH or vehicle injections. Rats were then killed. Sera, femora, tibiae, and kidneys were removed for chemical and histomorphometric analyses. PTH, PTH-CT3, and PTH-CT12 rats showed significant increases in total bone calcium (18-23%), dry weight (DW, 13-25%), and bone-forming surfaces compared with their respective controls. Eroded (resorption) surfaces were comparable between the groups. Although weight gain and serum calcium were normal in rats treated for 3 days with CT, rats treated for 12 days with CT gained 14% less weight than controls and were hypophosphatemic, with reduced serum calcium and urea nitrogen. Total bone mass increased both in Cl2MDP rats (Ca 21%, DW 2%), where resorption was presumably blocked, and in PTH rats (Ca 31%, DW 19%). The increase in bone mass was greater in PTH-Cl2MDP rats (Ca 48%, DW 29%) than in rats treated with Cl2MDP alone, suggesting that although Cl2MDP blocked resorption, the anabolic response to PTH was not altered. As neither short-term treatment with CT nor Cl2MDP blocked the anabolic response of bone to hPTH-(1-34), this response does not appear to depend on the early stimulation of resorption.

Anabolic effects of human biosynthetic parathyroid hormone fragment (1-34), LY333334, on remodeling and mechanical properties of cortical bone in rabbits.

Intermittent administration of parathyroid hormone (PTH) has an anabolic effect in cancellous bone of osteoporotic humans. However, the effect of PTH on cortical bone with Haversian remodeling remains controversial. The aim of this study was to determine the effects of biosynthetic human PTH(1-34) on the histology and mechanical properties of cortical bone in rabbits, which exhibit Haversian remodeling. Mature New Zealand white rabbits were treated with once daily injections of vehicle, or PTH(1-34), LY333334, at 10 micrograms/kg/day or 40 micrograms/kg/day for 140 days. Body weight in rabbits treated with PTH did not change significantly over the experimental period. Serum calcium and phosphate were within the normal range, but a 1 mg/ml increase in serum calcium was observed in rabbits given the higher dose of PTH. Histomorphometry of cortical bone in the midshaft of the tibia showed significant increases in periosteal and endocortical bone formation in these rabbits. Intracortical bone remodeling in the tibia was activated and cortical porosity increased by PTH. Cross-sectional bone area and bone mass of the midshaft of the femur increased significantly after PTH treatment. Ultimate force, stiffness, and work to failure of the midshaft of the femur of rabbits given the 40 micrograms dose of PTH were significantly greater than those in the control group, whereas elastic modulus was significantly lower than that in the rabbits given the 10 micrograms dose of PTH, but not different from controls. In the third lumbar vertebra, PTH increased both formation and resorption without increasing cancellous bone volume. The increases in bone turnover and cortical porosity were accompanied by concurrent increases in bone at the periosteal and endocortical surfaces. The combination of these phenomena resulted in an enhancement of the ultimate stress, stiffness, and work to failure of the femur.

Intermittently administered human parathyroid hormone(1-34) treatment increases intracortical bone turnover and porosity without reducing bone strength in the humerus of ovariectomized cynomolgus monkeys.

Cortical porosity in patients with hyperparathyroidism has raised the concern that intermittent parathyroid hormone (PTH) given to treat osteoporotic patients may weaken cortical bone by increasing its porosity. We hypothesized that treatment of ovariectomized (OVX) cynomolgus monkeys for up to 18 months with recombinant human PTH(1-34) [hPTH(1-34)] LY333334 would significantly increase porosity in the midshaft of the humerus but would not have a significant effect on the strength or stiffness of the humerus. We also hypothesized that withdrawal of PTH for 6 months after a 12-month treatment period would return porosity to control OVX values. OVX female cynomolgus monkeys were given once daily subcutaneous (sc) injections of recombinant hPTH(1-34) LY333334 at 1.0 microg/kg (PTH1), 5.0 microg/kg (PTH5), or 0.1 ml/kg per day of phosphate-buffered saline (OVX). Sham OVX animals (sham) were also given vehicle. After 12 months, PTH treatment was withdrawn from half of the monkeys in each treatment group (PTH1-W and PTH5-W), and they were treated for the remaining 6 months with vehicle. Double calcein labels were given before death at 18 months. After death, static and dynamic histomorphometric measurements were made intracortically and on periosteal and endocortical surfaces of sections from the middiaphysis of the left humerus. Bone mechanical properties were measured in the right humeral middiaphysis. PTH dose dependently increased intracortical porosity. However, the increased porosity did not have a significant detrimental effect on the mechanical properties of the bone. Most porosity was concentrated near the endocortical surface where its mechanical effect is small. In PTH5 monkeys, cortical area (Ct.Ar) and cortical thickness (Ct.Th) increased because of a significantly increased endocortical mineralizing surface. After withdrawal of treatment, porosity in PTH1-W animals declined to sham values, but porosity in PTH5-W animals remained significantly elevated compared with OVX and sham. We conclude that intermittently administered PTH(1-34) increases intracortical porosity in a dose-dependent manner but does not reduce the strength or stiffness of cortical bone.

Osteoblast apoptosis and bone turnover.

With the discoveries of different death mechanisms, an emerging definition of apoptosis is the process of cell death associated with caspase activation or caspase-mediated cell death. This definition accepts that caspases represent the final common mechanistic pathway in apoptosis. Apoptosis may be triggered either by activation events that target mitochondria or endoplasmic reticulum or by activation of cell surface "death receptors," for example, those in the tumor necrosis factor (TNF) superfamily. In the postnatal and adult skeleton, apoptosis is integral to physiological bone turnover, repair, and regeneration. The balance of osteoblast proliferation, differentiation, and apoptosis determines the size of the osteoblast population at any given time. Although apoptosis has been recorded in many studies of bone, the selective mechanisms invoked in the different models studied rarely have been identified. This review offers a broad overview of the current general concepts and controversies in apoptosis research and then considers specific examples of osteoblast apoptosis pertinent to skeletal development and to the regulation of bone turnover. In reviewing selected work on interdigital apoptosis in the developing skeleton, we discuss the putative roles of the bone morphogenetic proteins (BMPs), Msx2, RAR-gamma, and death inducer obliterator 1 (DIO-1). In reviewing factors regulating apoptosis in the postnatal skeleton, we discuss roles of cytokines, growth factors, members of the TNF pathway, and the extracellular matrix (ECM). Finally, the paradoxical effects of parathyroid hormone (PTH) on osteoblast apoptosis in vivo are considered in the perspective of a recent hypothesis speculating that this may be a key mechanism to explain the anabolic effects of the hormone. An improved understanding of the apoptotic pathways and their functional outcomes in bone turnover and fracture healing may facilitate development of more targeted therapeutics to control bone balance in patients with osteoporosis and other skeletal diseases.

Six-month daily administration of parathyroid hormone and parathyroid hormone-related protein peptides to adult ovariectomized rats markedly enhances bone mass and biomechanical properties: a comparison of human parathyroid hormone 1-34, parathyroid hormone-related protein 1-36, and SDZ-parathyroid hormone 893.

Daily administration of parathyroid hormone (PTH) and PTH-related protein (PTHrP) peptides has been shown to increase bone mass and strength in animals and, for PTH, to increase bone mass in humans. Long-term direct comparison of multiple members of the PTH/PTHrP family in vivo has not been reported. We therefore selected three PTH/PTHrP molecules for direct comparison in vivo in an adult rat model of postmenopausal osteoporosis: PTH(1-34), PTHrP(1-36), and the PTH analog, SDZ-PTH 893 ¿Leu8, Asp10, Lys11, Ala16, Gln18, Thr33, Ala34 human PTH 1-34 [hPTH(1-34)]¿. A 6-month study was performed in which adult (6-month-old) vehicle-treated ovariectomized (OVX) and sham OVX rats were compared with OVX rats receiving 40 micrograms/kg per day of either PTH(1-34), PTHrP(1-36), or PTH-SDZ-893. Bone mass, as assessed by ash weight and densitometry, bone histomorphometry, biomechanical properties at trabecular and cortical sites, and indices of bone formation markedly increased in all three PTH/PTHrP peptide-treated groups as compared with controls. In general, this improvement followed a rank order of SDZ-PTH-893 > PTH > PTHrP. The adverse effect profile also was greatest with SDZ-PTH-893; these rats developed moderate hypercalcemia, marked renal calcium accumulation, and displayed a 13% mortality. These studies show that PTH(1-34), PTHrP(1-36), and PTH-SDZ-893 significantly and progressively increase bone mass and bone strength in this rat model of postmenopausal osteoporosis. The adverse effect profile correlates in general terms with efficacy. All three peptides show promise as skeletal anabolic agents. Further studies in humans will be required to define optimal efficacy-to-adverse effect ratios and relative efficacy for each peptide in human osteoporosis.

The addition of a raloxifene analog (LY117018) allows for reduced PTH(1-34) dosing during reversal of osteopenia in ovariectomized rats.

To test the hypothesis that an antiresorptive agent might reduce the dosing requirement for an anabolic drug during reversal of osteopenia due to estrogen deficiency, the following experiment was conducted in 6-month-old female rats. Ovariectomy or sham surgery was performed and the following six experimental groups were studied. Untreated (SHAM) or ovariectomized (OVX) animals served as control groups. Four weeks post-OVX, osteopenic rats (now 7 months old), were treated in one of four experimental protocols: human parathyroid hormone (hPTH(1-34)), 80 microg/kg/day, given by subcutaneous injection 5 days/week; a selective estrogen receptor modulator (SERM), raloxifene analog LY117018 (RA), 3 mg/kg/day, given by gavage 5 days/week; and two combinations of LY117018 at the same dose and frequency with hPTH(1-34) (same dose, 5 times/week) and a reduced dosing interval of hPTH(1-34) (same dose, 2 times/week). After 12 weeks of treatment, the four experimental groups were sacrificed at age 10 months. SHAM and OVX controls were also studied at 7 and 10 months of age. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry at four skeletal sites: two mixed cortical/trabecular sites (femur and tibia) and two predominantly trabecular sites (lumbar spine and pelvis). The differences in BMD were consistent at all four sites. RA alone maintained BMD at all skeletal sites, but the results were not significantly improved over OVX controls, at age 10 months. hPTH(1-34) injections given 5 days/week resulted in BMD increments significantly higher than in either OVX or SHAM controls (p < 0.001). While the RA did not enhance the anabolic effects of full doses of hPTH(1-34), the addition of RA treatment to twice-weekly hPTH(1-34) dosing resulted in BMD increments at all four skeletal sites that were similar to the more intensive anabolic regimen of hPTH(1-34) therapy given 5 times/week. Therefore, an antiresorptive agent such as SERMs may potentially reduce the pharmacologic doses of PTH needed to reverse estrogen deficiency-induced osteopenia.

Parathyroid hormone regulates the expression of fibroblast growth factor-2 mRNA and fibroblast growth factor receptor mRNA in osteoblastic cells.

We examined the effect of parathyroid hormone (PTH) on basic fibroblast growth factor-2 (FGF-2) and FGF receptor (FGFR) expression in osteoblastic MC3T3-E1 cells and in neonatal mouse calvariae. Treatment of MC3T3-E1 cells with PTH(1-34) (10-8M) or forskolin (FSK; 10-5M) transiently increased a 7 kb FGF-2 transcript with a peak at 2 h. The PTH increase in FGF-2 mRNA was maintained in the presence of cycloheximide. PTH also increased FGFR-1 mRNA at 2 h and transiently increased FGFR-2 mRNA at 1 h. FGFR-3 and FGFR-4 mRNA transcripts were not detected in MC3T3-E1 cells. In cells transiently transfected with an 1800-bp FGF-2 promoter-luciferase reporter, PTH and FSK increased luciferase activity at 2 h and 4 h. Immunohistochemistry showed that PTH and FSK increased FGF-2 protein labeling in the nuclei of MC3T3-E1 cells. PTH also increased FGF-2 mRNA, and FGFR-1 and FGFR-2 mRNA levels within 30 minutes in neonatal mouse calvarial organ cultures. We conclude that PTH and cAMP stimulate FGF-2 mRNA abundance in part through a transcriptional mechanism. PTH also regulated FGFR gene expression. We hypothesize that some effects of PTH on bone remodeling may be mediated by regulation of FGF-2 and FGFR expression in osteoblastic cells.

Comparison of recombinant human PTH(1-34) (LY333334) with a C-terminally substituted analog of human PTH-related protein(1-34) (RS-66271): In vitro activity and in vivo pharmacological effects in rats.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are believed to exert their biological actions through binding and activation of a common cell surface receptor. Recently, an analog of PTHrP (RS-66271), was described that demonstrated reduced binding affinity for the PTH/PTHrP receptor compared with bovine PTH(1-34) but retained equal biological activity. The present study investigated the receptor binding affinities of synthetic RS-66271 and recombinant human PTH(1-34) (LY333334) and compared their in vitro and in vivo pharmacological effects. RS-66271 had one hundredth the activity of PTH(1-34) in competing for the binding of [125I] [Nle8,18, Tyr34]human PTH(1-34) to the human PTH/PTHrP receptor stably expressed in a human kidney cell line. Despite this reduced binding affinity, RS-66271 had equivalent activity in increasing both cAMP production in osteoblast-like cells and bone resorption in neonatal mouse calvariae. However, RS-66271 was 7. 6-fold less active in stimulating inositol phosphate production. For in vivo studies, young, male Fisher rats received a daily subcutaneous dose of either 10 or 40 microg/kg of peptide for 1, 2, or 4 weeks. Volumetric bone mineral density and total bone mineral content of the proximal tibia were determined by peripheral quantitative computerized tomography. Trabecular and cortical bone of the distal femur were analyzed for calcium and dry weight. Lumbar vertebrae (L4-L6) were analyzed by histomorphometry. Trabecular and cortical bone mass showed a dose- and time-dependent increase in the treated animals compared with the controls. These increases were evident as early as 1 week after initiation of dosing. There were no consistent significant differences in the comparative effects of PTH(1-34) and RS-66271 on the measured bone parameters. In conclusion, despite the reduced binding affinity of RS-66271 for the PTH/PTHrP receptor compared with human PTH(1-34), both peptides displayed similar in vitro and in vivo pharmacological effects.

Cloning and functional analysis of a family of nuclear matrix transcription factors (NP/NMP4) that regulate type I collagen expression in osteoblasts.

Collagen expression is coupled to cell structure in connective tissue. We propose that nuclear matrix architectural transcription factors link cell shape with collagen promoter geometry and activity. We previously indicated that nuclear matrix proteins (NP/NMP4) interact with the rat type I collagen alpha1(I) polypeptide chain (COL1A1) promoter at two poly(dT) sequences (sites A and B) and bend the DNA. Here, our objective was to determine whether NP/NMP4-COL1A1 binding influences promoter activity and to clone NP/NMP4. Promoter-reporter constructs containing 3.5 kilobases (kb) of COL1A1 5' flanking sequence were fused to a reporter gene. Mutation of site A or site B increased promoter activity in rat UMR-106 osteoblast-like cells. Several full-length complementary DNAs (cDNAs) were isolated from an expression library using site B as a probe. These clones expressed proteins with molecular weights and COLIA1 binding activity similar to NP/NMP4. Antibodies to these proteins disrupted native NP/NMP4-COL1A1 binding activity. Overexpression of specific clones in UMR-106 cells repressed COL1A1 promoter activity. The isolated cDNAs encode isoforms of Cys2His2 zinc finger proteins that contain an AT-hook, a motif found in architectural transcription factors. Some of these isoforms recently have been identified as Cas-interacting zinc finger proteins (CIZ) that localize to fibroblast focal adhesions and enhance metalloproteinase gene expression. We observed NP/NMP4/CIZ expression in osteocytes, osteoblasts, and chondrocytes in rat bone. We conclude that NP/NMP4/CIZ is a novel family of nuclear matrix transcription factors that may be part of a general mechanical pathway that couples cell structure and function during extracellular matrix remodeling.

Parenteral pamidronate prevents thyroid hormone-induced bone loss in rats.

Pamidronate (APD) is a bisphosphonate that prevents bone loss from a variety of causes. We studied the role of APD in preventing thyroid hormone-induced bone loss. A total of 32 rats were assigned to one of four treatment groups: (1) -APD/triiodothyronine (-T3), (2) -APD/+T3, (3) +APD/-T3, or (4) +APD/+T3. In the first of two studies, the rats received APD for the first week and T3 for the second week, and then their blood was analyzed for alkaline phosphatase and osteocalcin. Alkaline phosphatase and osteocalcin were significantly higher (p < 0.05) in hyperthyroid rats (-APD/+T3, 3.9 +/- 0.25 mukat/liter and 23 +/- 1.6 nM, respectively) than in control animals (2.53 +/- 0.28 mukat/liter and 18.3 +/- 1.4 nM, respectively). Hyperthyroid rats pretreated with APD (+APD/+T3) had levels of alkaline phosphatase and osteocalcin no different from controls. In a second study, rats were divided into the same four groups, except they received APD/placebo and T3/placebo concomitantly for 3 weeks. At the end of the study, bone mineral density (BMD) of the femur, spine, and whole body was measured by dual-energy x-ray absorptiometry, and the calcium content of the femora was measured directly. In hyperthyroid rats (-APD/+T3) BMD was significantly lower than in controls in the spine (0.201 +/- 0.004 versus 0.214 +/- 0.002 g/cm2, p < 0.05) and femur (0.204 +/- 0.003 versus 0.218 +/- 0.002, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)