Effects of calcium depletion and repletion on serum insulin-like growth factor I and binding protein levels in weanling rats.

Previous studies in a weanling rat model indicated that dietary calcium depletion not only stimulated osteoclastic resorption but also inhibited bone formation. The present study sought to test whether the depletion-associated inhibition of bone formation is related to a reduction in serum insulin-like growth factor-I (IGF-I) and/or an increase in its binding proteins (IGFBPs). Twenty male weanling rats were divided into two weight-matched groups.

Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice.

Previous studies have shown that C3H/HeJ (C3H) mice have higher peak bone density than C57BL/6J (B6) mice, at least in part because of differences in rates of bone resorption. The current studies were intended to examine the alternative, additional hypothesis that the greater bone density in C3H mice might also be a consequence of increased bone formation. To that end, we measured two presumptive, indirect indices of bone formation and osteoblast number in these inbred strains of mice: alkaline phosphatase (ALP) activity in serum, bones, and bone cells; and the number of ALP-positive colony-forming units (CFU) in bone marrow stromal cell cultures.

Effects of inhibitors of signal transduction pathways on transforming growth factor beta1 and osteogenic protein-1-induced insulinlike growth factor binding protein-3 expression in human bone cells.

Signal transduction initiated by TGFbeta1 and OP-1 was studied in MG63 human osteosarcoma cells and in normal human bone cells (HBCs) in the presence of inhibitors of signal transduction events, using insulinlike growth factor binding protein-3 (IGFBP-3) production as an end point. Treatment of serum-free MG63 cells and normal HBCs with TGFbeta1 increased IGFBP-3 protein level several fold in the conditioned medium. This effect of TGFbeta1 was mediated by increased de novo synthesis because mRNA level increased to the same extent as protein level and TGFbeta1 treatment had very little effect on IGFBP-3 protease activity.

Osteogenic protein-1 stimulates production of insulin-like growth factor binding protein-3 nuclear transcripts in human osteosarcoma cells.

To begin delineating molecular mechanisms by which osteogenic protein-1 (OP-1) modulates its effect on the insulin-like growth factor (IGF) system in human skeletal cells, we evaluated time-course effects of OP-1 on the expression of IGFBP-3 messenger RNA (mRNA) in human SaOS-2 osteosarcoma cells and found that 100 ng/ml of OP-1 increased (maximum 10.7-fold at 24 h; P < 0.01) the level of IGFBP-3 mRNA in a time-dependent manner (from 3-36 h; treatment x time interaction, P < 0.

Circulating and skeletal insulin-like growth factor-I (IGF-I) concentrations in two inbred strains of mice with different bone mineral densities.

Recent work has demonstrated differences in femoral bone mineral density between two common inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), across a wide age range. To investigate one possible mechanism that could affect acquisition and maintenance of bone mass in mice, we studied circulatory and skeletal insulin-like growth factor-I (IGF-I) and femoral bone mineral density (F-BMD) by pQCT in C3H and B6 progenitor strains, as well as serum IGF-I obtained from matings between these two strains and mice bred from subsequent F1 intercrosses (F2). Serum IGF-I measured by radioimmunoassay was more than 35% higher in virgin progenitor C3H than virgin B6 at 1, 4, 8, and 10 months of age, and in 8-month-old C3H compared with B6 retired breeders (p < 0.

Serum insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5 levels in aging and age-associated diseases.

Because impaired bone formation is a major contributor to the pathogenesis of senile (type II) osteoporosis, and because insulin-like growth factors (IGFs) have been shown to be important stimulators of bone formation in vitro and in vivo, studies have been focused towards clarifying the role of IGF system in the age-related impairment in bone formation. To evaluate if changes in circulating and bone cell production of IGF binding protein (IGFBP)-4 and IGFBP-5 could contribute to age-related impairment in bone formation, the authors measured serum and skeletal levels of IGFBP-5, and serum levels of IGFBP-4 during aging. Circulating levels of IGFBP-4 increased with age, whereas that of IGFBP-5 declined with age.

Fluoride at mitogenic doses induces a sustained activation of p44mapk, but not p42mapk, in human TE85 osteosarcoma cells.

Fluoride, at micromolar concentrations, stimulates bone cell proliferation in vitro. In this study, we sought to test whether fluoride at mitogenic doses increases the tyrosyl phosphorylation level and specific activity of a mitogen-activated protein kinase (MAPK) in human TE85 osteosarcoma cells. Analysis by immunoprecipitation with antiphosphotyrosine antibody followed by Western analysis using an anti-pan extracellular signal-regulated kinase antibody revealed that fluoride at the optimal mitogenic dose (i.

Insulin-like growth factor (IGF)-I, -II, IGF binding proteins (IGFBP)-3, -4, and -5 levels in the conditioned media of normal human bone cells are skeletal site-dependent.

The skeleton in its function of affording strength and support to the body is subject to differential mechanical loading which has been implicated to mediate some of its effects on bone formation via the insulin-like growth factors (IGFs), which are important regulators of bone metabolism. We, therefore, sought to conduct the present study with the hypothesis that the skeletal site-dependent differences in mechanical loading and other variables including stage of osteoblast differentiation would be associated with site-specific differences in the production of the IGF system components. To test this hypothesis, conditioned media (CM) from normal human bone cells (control and IGF-II-treated 48-h cultures) from five different skeletal sites were obtained and assayed for IGF-I, IGF-II (following separation of IGF binding proteins [IGFBPs]), IGFBP-3, IGFBP-4, and IGFBP-5 protein levels employing specific radioimmunoassays for each protein.

Osteogenic protein-1 stimulates mRNA levels of BMP-6 and decreases mRNA levels of BMP-2 and -4 in human osteosarcoma cells.

Bone morphogenetic proteins (BMPs) are novel growth and differentiation factors that act on mesenchymal stem cells to initiate new bone formation in vivo and promote the growth and differentiation of cells in the osteoblastic lineage. In the present study, we examined the effects of recombinant human osteogenic protein-1 (also known as BMP-7) on the expression of related members of the BMP family using SaOS-2 and U2-OS, two human osteosarcoma cell strains. Evaluation of BMP-2, -4, and -6 mRNA expression indicates that OP-1 stimulated the mRNA levels of BMP-6 in both SaOS-2 cells (threefold) and U2-OS cells (fivefold) after 24 hours of treatment, while decreasing the mRNA levels of BMP-4 in SaOS-2 cells (80%) and BMP-2 and BMP-4 in U2-OS cells by 50% and 72%, respectively.

Structural and functional analysis of the 5'-flanking region of the human insulin-like growth factor binding protein (IGFBP)-4 gene.

More than 3 kb of the human (h)IGFBP-4 gene 5'-flanking region was sequenced and assessed for promoter activity. The hIGFBP-4 promoter resides within a CpG island and demonstrates strong basal activity in human osteoblast-like osteosarcoma and COS-7 monkey kidney cells. Transient transfection of cells with hIGFBP-4 promoter-linked deletion constructs demonstrated that multiple cooperating cis-acting elements within 836 bp of the 5'-flanking region contributed to overall promoter strength.

Serum fluoride and serum osteocalcin levels in response to a novel sustained-release monofluorophosphate preparation: comparison with plain monofluorophosphate.

In a previous study we found that sustained-release monofluorophosphate (MFP-SR), a novel, sustained-release MFP preparation, acutely maintained the basal therapeutic serum fluoride levels without causing the high serum peak levels associated with plain MFP administration. The objective of the present study was to determine (a) whether chronic MFP-SR administration would provide therapeutic serum fluoride levels, and (b) whether treatment with this new preparation would result in an increase in bone formation similar to that achieved with plain MFP. Bone formation was assessed by serum osteocalcin (OC) determination.

Osteogenic actions of phenytoin in human bone cells are mediated in part by TGF-beta 1.

We have recently demonstrated that phenytoin, a widely used therapeutic agent for seizure disorders, has osteogenic effects in rats and in humans in vivo, and in human bone cells in vitro. The goal of the present study was to determine the mechanism of the osteogenic action of phenytoin in normal human mandible-derived bone cells. Because many osteogenic agents increased bone cell proliferation through mediation by growth factors, we tested the hypothesis that the osteogenic effects of phenytoin involved the release of a growth factor by measuring the mRNA level of several bone cell growth factors and insulin-like growth factor (IGF) binding proteins with Northern blots using specific cDNA probes.

Truncation of the amino terminus of PTH alters its anabolic activity on bone in vivo.

In vitro studies of parathyroid hormone (PTH) structure and function have suggested that the anabolic effect of PTH on bone requires the presence of amino acid residues 28-34 (domains for protein kinase C activation and mitogenic activity), but not amino acid residues 1-7 (adenylate cyclase activation domain). We have tested this hypothesis with in vivo studies of human PTH (hPTH) analogs. Serum biomarkers and selected histomorphometric parameters of bone formation and resorption were assessed in adult, female, Sprague-Dawley rats following 19 daily injections of vehicle, 10 micrograms/kg body weight (bw) of hPTH(1-38), or a dose range of 10, 40, and 100 micrograms/100 g bw of hPTH(2-38) or hPTH(3-38).

Evaluation of signal transduction mechanisms for the mitogenic effects of prostaglandin E2 in normal human bone cells in vitro.

Prostaglandin E2 (PGE2) is one of the most potent stimulators of bone formation in vivo. In these studies, we investigated the mechanism(s) underlying PGE2 effects on human bone formation by evaluating the effects of PGE2 on normal human bone cell (HBC) proliferation in vitro. Cell proliferation of normal HBCs was increased by PGE2 as measured by increased [3H]thymidine incorporation after 18 h and increased cell number after 48 h of treatment.

Growth factors for bone growth and repair: IGF, TGF beta and BMP.

Current research is reviewed regarding the actions of three growth factor systems on bone formation: insulin-like growth factors (IGFs), transforming growth factor-betas (TGF betas), and bone morphogenetic proteins (BMPs). Each growth factor family consists of multiple related growth factor genes. TGF betas and BMPs 2-7 are subfamilies of a larger TGF beta superfamily.

Fluoride at mitogenic concentrations increases the steady state phosphotyrosyl phosphorylation level of cellular proteins in human bone cells.

This study was designed to test the hypothesis that treatment of human bone cells with mitogenic concentrations of fluoride would lead to an increase in the steady state level of tyrosyl phosphorylation of specific cellular proteins. With an immunoblot assay method, it was found that mitogenic concentrations of fluoride (i.e.

Growth hormone (GH) treatment of GH-deficient children increases serum levels of insulin-like growth factors (IGFs), IGF-binding protein-3 and -5, and bone alkaline phosphatase isoenzyme.

To investigate the contribution of the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) to the regulation of bone growth in 10 GH-deficient Japanese children receiving recombinant GH therapy, we determined the percent increase from pretreatment levels of serum IGF-I, IGF-II, IGFBP-3, IGFBP-5, and bone-specific alkaline phosphatase isoenzyme (B-ALP). For 10 children between 6-13 yr of age, serum IGF-I and IGF-II were increased after 1 month of treatment by 53% and 7%, respectively; after 12 months of therapy, IGF levels remained elevated at 51% and 17%, respectively. Serum IGFBP-3 and IGFBP-5 were also increased after 1 month of GH therapy by 17% and 13% respectively; after 12 months of therapy, they remained elevated at 22% and 15%, respectively.

Molecular cloning and expression of a unique rabbit osteoclastic phosphotyrosyl phosphatase.

Tyrosyl phosphorylation plays an important regulatory role in osteoclast formation and activity. Phosphotyrosyl phosphatases (PTPs), in addition to tyrosyl kinases, are key determinants of intracellular tyrosyl phosphorylation levels. To identify the PTP that might play an important regulatory role in osteoclasts, we sought to clone an osteoclast-specific PTP.

Genetic variability in adult bone density among inbred strains of mice.

More than 70% of the variability in human bone density has been attributed to genetic factors as a result of studies with twins, osteoporotic families, and individuals with rare heritable bone disorders. We have applied the Stratec XCT 960M pQCT, specifically modified for small skeletal specimens, to analyses of bones from 11 inbred strains (AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, NZB/B1NJ, SM/J, SJL/BmJ, SWR/BmJ, and 129/J) of female mice to determine the extent of heritable differences in peak bone density, pQCT scans were taken of femurs from (a) 12-month-old inbred strain females and (b) a subset of four strains (C3H/HeJ, DBA/2J, BALB/cByJ, C57BL/6J) at 2, 4, and 8 months. In addition, pQCT scans were also obtained from L5-L6 vertebrae and proximal phalanges from the same subset of four inbred strains at 12 months of age.

Insulin-like growth factor-binding protein-6 produced by human PC-3 prostate cancer cells: isolation, characterization and its biological action.

The PC-3 human prostatic carcinoma cell line has been extensively used as a model for studies on the regulation of prostate tumor cell proliferation. Because of the importance of IGF-binding proteins (IGFBPs) in the control of IGF activities that regulate cell proliferation in normal and malignant cell types, we undertook studies to characterize the IGFBPs produced by PC-3 prostate tumor cells in culture. We previously found, using an IGF-I affinity column for purification and a polyethylene glycol (PEG) precipitation assay for IGFBP detection, that PC-3 cells in culture produced a single predominant IGFBP, IGFBP-4, which inhibits IGF activities.

Evidence for a role for insulin-like growth factor binding proteins in glucocorticoid inhibition of normal human osteoblast-like cell proliferation.

Glucocorticoids (GCs) inhibit bone formation in vivo and inhibit osteoblast proliferation and collagen synthesis in vitro. These effects may be mediated by alterations in the insulin-like growth factor (IGF) system. In the present study of normal human osteoblast-like (HOB) cells, we tested the hypothesis that dexamethasone (Dex) inhibits IGF anabolic activity in bone by altering expression of IGF binding proteins (IGFBPs), particularly by decreasing expression of IGFBP-5 and IGFBP-3 (which enhance IGF activity) and increasing expression of IGFBP-4 (which inhibits IGF actions).