Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells.

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.

Transcriptional induction of prostaglandin G/H synthase-2 by basic fibroblast growth factor.

In serum-free mouse osteoblastic MC3T3-E1 cells, basic fibroblastic growth factor (bFGF) induced mRNA and protein for prostaglandin G/H synthase-2 (PGHS-2), the major enzyme in arachidonic acid (AA) conversion to prostaglandins. mRNA accumulation peaked at 1 h with bFGF 1 nM. In cells stably transfected with a 371-bp PGHS-2 promoter-luciferase reporter, bFGF stimulated luciferase activity, which peaked at 2-3 h with bFGF 1-10 nM. In the presence of exogenous AA, bFGF stimulated PGE2 production, which paralleled luciferase activity. In serum-free neonatal mouse calvarial cultures, bFGF stimulated PGE2 production in the absence of exogenous AA. bFGF stimulated PGHS-2 mRNA accumulation, which peaked at 2-4 h and then decreased; there were later mRNA elevations at 48 and 96 h that were inhibited by indomethacin. In both MC3T3-E1 cells and neonatal calvariae, bFGF produced smaller and slower increases in PGHS-1 mRNA levels than for PGHS-2. bFGF stimulated bone resorption in mouse calvariae with a maximal increase of 80% at 1 nM. Stimulation was partially inhibited by nonsteroidal anti-inflammatory drugs. We conclude that bFGF rapidly stimulates PGE2 production in osteoblasts, largely through transcriptional regulation of PGHS-2, and that prostaglandins mediate some of bFGF's effects on bone resorption.

Primary hyperparathyroidism caused by parathyroid-targeted overexpression of cyclin D1 in transgenic mice.

The relationship between abnormal cell proliferation and aberrant control of hormonal secretion is a fundamental and poorly understood issue in endocrine cell neoplasia. Transgenic mice with parathyroid-targeted overexpression of the cyclin D1 oncogene, modeling a gene rearrangement found in human tumors, were created to determine whether a primary defect in this cell-cycle regulator can cause an abnormal relationship between serum calcium and parathyroid hormone response, as is typical of human primary hyperparathyroidism. We also sought to develop an animal model of hyperparathyroidism and to examine directly cyclin D1's role in parathyroid tumorigenesis. Parathyroid hormone gene regulatory region--cyclin D1 (PTH--cyclin D1) mice not only developed abnormal parathyroid cell proliferation, but also developed chronic biochemical hyperparathyroidism with characteristic abnormalities in bone and, notably, a shift in the relationship between serum calcium and PTH. Thus, this animal model of human primary hyperparathyroidism provides direct experimental evidence that overexpression of the cyclin D1 oncogene can drive excessive parathyroid cell proliferation and that this proliferative defect need not occur solely as a downstream consequence of a defect in parathyroid hormone secretory control by serum calcium, as had been hypothesized. Instead, primary deregulation of cell-growth pathways can cause both the hypercellularity and abnormal control of hormonal secretion that are almost inevitably linked together in this common disorder.

Biomimetic calcium phosphate/polyelectrolyte multilayer coatings for sequential delivery of multiple biological factors.

Combinations of growth factors synergistically enhance tissue regeneration, but typically require sequential, rather than co-delivery from biomaterials for maximum efficacy. Polyelectrolyte multilayer (PEM) coatings can deliver multiple factors without loss of activity; however, sequential delivery from PEM has been limited due to interlayer diffusion that results in co-delivery of the factors. This study shows that addition of a biomimetic calcium phosphate (bCaP) barrier layer to a PEM coating effectively prevents interlayer diffusion and enables sequential delivery of two different biomolecules via direct cell access. A simulated body fluid method was used to deposit a layer of bCaP followed by 30 bilayers of PEM made with poly-l-Lysine (+) and poly l-Glutamic acid (-) (bCaP-PEM). Measurements of MC3T3-E1 proliferation and viability over time on bCaP-PEM were used to demonstrate the sequential delivery kinetics of a proliferative factor [fibroblast growth factor-2 (FGF-2)] followed by a cytotoxic factor (antimycin A, AntiA). FGF-2 and AntiA both retained their bioactivity within bCaP-PEM, yet no release of FGF-2 or AntiA from bCaP-PEM was observed when cells were absent indicating a cell-mediated, local delivery process. This coating technique is useful for a variety of applications that would benefit from highly localized, sequential delivery of multiple biomolecules governed by cell initiated degradation that avoids off-target effects associated with diffusion-based release. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1500-1509, 2017.

Congenic mice provide in vivo evidence for a genetic locus that modulates serum insulin-like growth factor-I and bone acquisition.

We identified quantitative trait loci (QTL) that determined the genetic variance in serum IGF-I through genome-wide scanning of mice derived from C57BL/6J(B6) x C3H/HeJ(C3H) intercrosses. One QTL (Igf1s2), on mouse chromosome 10 (Chr10), produces a 15% increase in serum IGF-I in B6C3 F2 mice carrying c3 alleles at that position. We constructed a congenic mouse, B6.C3H-10 (10T), by backcrossing c3 alleles from this 57-Mb region into B6 for 10 generations. 10T mice have higher serum and skeletal IGF-I, greater trabecular bone volume fraction, more trabeculae, and a higher number of osteoclasts at 16 wk, compared with B6 (P < 0.05). Nested congenic sublines generated from further backcrossing of 10T allowed for recombination and produced four smaller sublines with significantly increased serum IGF-I at 16 wk (i.e. 10-4, 10-7, 10-10, and 10-13), compared with B6 (P < 0.0003), and three smaller sublines that showed no differences in IGF-I vs. age- and gender-matched B6 mice. Like 10T, the 10-4 nested sublines at 16 wk had higher femoral mineral (P < 0.0001) and greater trabecular connectivity density with significantly more trabeculae than B6 (P < 0.01). Thus, by comprehensive phenotyping, we were able to narrow the QTL to an 18.3-Mb region containing approximately 148 genes, including Igf1 and Elk-3(ETS domain protein). Allelic differences in the Igf1s2 QTL produce a phenotype characterized by increased serum IGF-I and greater peak bone density. Congenic mice establish proof of concept of shared genetic determinants for both circulating IGF-I and bone acquisition.

Synthetic peptide containing Arg-Gly-Asp inhibits bone formation and resorption in a mineralizing organ culture system of fetal rat parietal bones.

The role of integrins, cell surface receptors involved in cell adhesion to the matrix, was studied in a mineralizing organ culture system. Integrin-mediated cell attachment to matrix proteins has been shown to depend partially on the amino acid sequence Arg-Gly-Asp (RGD), present in the extracellular matrix proteins. Therefore, the effect of RGD peptides on bone formation and resorption was studied in the mineralizing organ culture system derived from 18 day fetal rat parietal bones. Addition of 0.1-50 microM GRGDSPK to bones cultured for 4 days inhibited mineralization in a dose-dependent manner as determined by measuring calcium content and % bone/unit area of tissue. A maximal decrease in calcium content and % bone/unit area of 32.5 and 42.9%, respectively, was found with 50 microM GRGDSPK. With 10 and 50 microM GRGDSPK, bone morphology was dramatically altered, with a disruption of osteoblast and mineralized matrix organization. To assess the effect of the peptides on bone resorption, fetal bones were prelabeled in vivo with 45Ca and resorption was stimulated in vitro with parathyroid hormone in the presence or absence of the peptide. A significant decrease in 45Ca release was found with 10 and 50 microM GRGDSPK. Osteoclast number was also significantly decreased on the bone surface. The peptide was not cytotoxic, since no effect on DNA content, dry weight, or collagen synthesis was found. GRADSP, a control peptide, had no significant effect on mineralization, resorption, or other parameters of bone growth.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of 1,25-dihydroxyvitamin D3 on the cytoskeleton of rat calvaria and rat osteosarcoma (ROS 17/2.8) osteoblastic cells.

1,25-dihydroxyvitamin D3 produces pronounced shape changes in fetal rat calvaria and osteosarcoma-derived (ROS 17/2.8) osteoblastic cells, characterized by retracting processes and cell rounding followed by aggregation of cells. The 1,25(OH)2D3 effect on ROS 17/2.8 morphology was determined morphometrically on scanning electron micrographs. The hormone effect was found to be dose dependent between 10(-12) and 10(-9) M. The shape changes appeared 12 h after hormone (10(-10) M) addition and were present in 80% of the ROS 17/2.8 cells and in 50% of the calvaria cells at 72 h. Cycloheximide at 1 microM, inhibited the hormone-dependent change in morphology. The 1,25(OH)2D3 effects were partially mimicked by 10(-8) M 25(OH)D3 but not by 10(-10) M 25(OH)D3 or 10(-11)-10(-8) M 24,25(OH)2D3. 1,25-dihydroxyvitamin D3 also increased cell proliferation twofold at 14 days in serum-free medium. 1,25(OH)2D3 treatment produced changes in microfilament organization, visualized with rhodamine-conjugated phalloidin. Microfilaments were localized at the terminal attachment points and in the perinuclear region, and few if any, were seen in the retracting processes themselves. Estimation of cytoskeletal actin and myosin by gel electrophoresis of Triton X-100 nonextractable proteins showed a 30% reduction in these proteins in the hormone-treated cells. Microtubules visualized by indirect immunofluorescence showed no major changes in organization. Both colchicine and cytochalasin D altered the hormone-induced shape change, suggesting that both microfilaments and microtubules were required for this process. Thus, 1,25(OH)2D3 had pronounced effects on cell shape in osteoblastic cells, probably via de novo protein synthesis. These changes lead to rearrangement of the cytoskeleton, primarily the microfilaments.

Glucocorticoids stimulate resorption in fetal rat parietal bones in vitro.

The effect of glucocorticoids on bone resorption was examined in a serum-free mineralizing organ culture system derived from 20 day fetal rat parietal bones. Bone resorption was assessed by prelabeling the fetal rats in utero with 45Ca and determining the daily release of 45Ca into the medium of cultured bones. During the first 24 h of treatment a transient stimulation of bone resorption was found; 4.5 +/- 0.3% of the total 45Ca was released into the medium with 1 nM corticosterone and 4.1 +/- 0.2% with 10 nM corticosterone compared to 2.9 +/- 0.2% in control bones. Treatment with 1 and 10 nM dexamethasone for 24 h also showed an increase in 45Ca release compared to control bones. During the same time period 45Ca release was 6.9 +/- 1.4% with 10 nM parathyroid hormone. At later time points 100 and 1000 nM corticosterone inhibited 45Ca release, but 1 and 10 nM corticosterone values were similar to controls. At 24 h the number of osteoclasts per mm2 tissue in bone lacunae was significantly elevated with 1-100 nM corticosterone and 10 nM parathyroid hormone compared to control bones. In control bones 0.10 +/- 0.05 osteoclasts per mm2 of tissue were found, but 0.59 +/- 0.21 osteoclasts per mm2 were seen with 10 nM corticosterone and 1.50 +/- 0.34 with 10 nM parathyroid hormone. An additional assay of bone resorption, the release of lysosomal beta-glucuronidase into the medium was also elevated in glucocorticoid and parathyroid hormone-treated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro mineralization of fetal rat parietal bones in defined serum-free medium: effect of beta-glycerol phosphate.

We have developed a bone organ culture system that mineralizes in vitro. Fetal rat parietal bones (20 days old) were cultured in a chemically defined serum-free medium containing physiological 3 mM phosphate. During 5 days in culture, calcium content increased from 26 to 55 micrograms and dry weight increased from 137 to 194 micrograms. After 2 days in vivo, the calcium content of the parietal bone showed a comparable increase to 49 micrograms and dry weight increased to 183 micrograms. During culture, the mineralized bone area in thick sections increased from 11 to 23%, which paralleled the doubling in calcium content. Fluorescent calcein labeling during the 5 day culture period demonstrated that calcification occurs in an ordered pattern. Protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP). The percentage collagen synthesis decreased from 17.5% at 0 time to 5.0% at 2 days and then increased to 9.4% at 5 days of culture. Varying the inorganic phosphate concentration in the medium or adding beta-glycerol phosphate was found to affect mineralization. After 5 days in culture, bones treated with 1 mM phosphate exhibited a large region of unmineralized osteoid with only a 23% increase in calcium content compared with 112% in control (3 mM phosphate) bones and a 28% increase in dry weight compared with a 40% increase in control. Treatment for 5 days with 6 mM phosphate or 1, 3, or 10 mM beta-glycerol phosphate had no significant effect on dry weight compared to control bones. However, bone calcium content increased significantly from 55 +/- 5 micrograms in control cultures to 105 +/- 7 with 6 mM phosphate, 74 +/- 6 with 3 mM beta-glycerol phosphate, and 75 +/- 5 micrograms with 10 mM beta-glycerol phosphate. Calcified area measured by histomorphometry was also significantly greater than in control bones, but this was mainly due to ectopic calcification in the periosteum, representing from 23 to 74% of the total increase in calcified matrix in bones cultured with 6 mM phosphate or 1-10 mM beta-glycerol phosphate. Ultrastructural analysis demonstrated that ectopic calcification was associated with cell death and debris. Therefore, calcification with beta-glycerol phosphate and high concentrations of inorganic phosphate differed from mineralization in vivo or in bones cultured with a physiologically concentration of phosphate.

Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage.

Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3-kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz-positive cells first appeared in spindle-shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz-positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.

Conditional ablation of the osteoblast lineage in Col2.3deltatk transgenic mice.

Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragment of the rat alpha1 type I collagen promoter driving a truncated form of the herpes thymidine kinase gene (Col2.3Atk). Expression of the transgene was found in osteoblasts coincident with other genetic markers of early osteoblast differentiation. Mice treated with ganciclovir (GCV) for 16 days displayed extensive destruction of the bone lining cells and decreased osteoclast number. In addition, a dramatic decrease in bone marrow elements was observed, which was more severe in the primary spongiosum and marrow adjacent to the diaphyseal endosteal bone. Immunostaining for transgene expression within the bone marrow was negative and marrow stromal cell cultures developed normally in the presence of GCV until the point of early osteoblast differentiation. Our findings suggest that the early differentiating osteoblasts are necessary for the maintenance of osteoclasts and hematopoiesis. Termination of GCV treatment produced an exaggerated response of new bone formation in cortical and trabecular bone. The Col2.3deltatk mouse should be a useful model to define the interrelation between bone and marrow elements as well as a model to analyze the molecular and cellular events associated with a defined wave of osteogenesis on termination of GCV treatment.

Regulation of NFIL-6 and IL-6 expression by basic fibroblast growth factor in osteoblasts.

We determined whether basic fibroblast growth factor (bFGF) regulated the expression of IL-6 and NFIL-6 in osteoblasts. In mouse osteoblastic MC3T3-E1 cells, bFGF (10(-8) M) increased NFIL-6 mRNA 2-fold at 30 minutes and 3-fold at 2 h. IL-6 mRNA was increased by bFGF 10(-8) M after 1 h. IL-6 protein was detectable in control cultures but was significantly increased by bFGF (10(-8) M) at 4 h. Immunofluorescence analysis of MC3T3-E1 cells showed primarily cytoplasmic and perinuclear NFIL-6 staining in control cultures while bFGF-treated cells showed increased NFIL-6 staining at 2 and 4 h. Western blotting revealed that bFGF increased NFIL-6 protein at 2 h. In calvarial mouse osteoblasts, bFGF 10(-8) M induced IL-6 mRNA as early as 1 h and significantly increased IL-6 protein levels as early as 2 h. In conclusion, bFGF stimulates IL-6 and NFIL-6 mRNA in osteoblasts. The increase in NFIL-6 mRNA was associated with increased NFIL-6 protein. The increase in IL-6 mRNA was also associated with increased IL-6 protein. We propose that activations of NFIL-6 and IL-6 may be important mediators of the effects of bFGF on bone cells.

Human parathyroid hormone 1-34 reverses bone loss in ovariectomized mice.

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss-Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1-34) [hPTH(1-34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (microCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5-week and 9-week post-OVX, non-PTH-treated animals. PTH induced a approximately 35% recovery of this loss and a approximately 40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein-labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2- and 3-fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss-Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.

Glucocorticoids inhibit the attachment of osteoblasts to bone extracellular matrix proteins and decrease beta 1-integrin levels.

Prolonged glucocorticoid treatment causes osteoporosis in vivo and inhibits bone formation in vitro. We have previously shown that glucocorticoids inhibit calcification and alter osteoblast organization in a mineralizing bone organ culture system. In this study, the effect of glucocorticoids on osteoblast adhesion to bone matrix proteins and integrin expression was examined in primary rat osteoblasts and a transformed rat osteosarcoma-derived cell line ROS 17/2.8. After 24 h of treatment with corticosterone, these cells displayed a concentration-dependent decrease in adhesion to type I collagen and fibronectin. Adhesion was significantly decreased as early as 4 h after glucocorticoid administration. With 100 nM corticosterone treatment for 24 h, inhibition of the adhesion of ROS 17/2.8 cells and primary osteoblasts to fibronectin was 75 +/- 10% and 50 +/- 8%, and inhibition of adhesion to collagen was 31 +/- 10% and 65 +/- 5%, respectively. This effect was specific for osteoblasts, because glucocorticoids did not change the adhesion of fibroblasts. However, glucocorticoids did inhibit the adhesion of all cell types to rat osteonectin. To determine whether the change in osteoblast attachment to collagen and fibronectin was due to an alteration in integrin levels, the plasma membranes of these cells were labeled with [125I]lactoperoxidase, solubilized, and immunoprecipitated with an antibody to beta 1. A 24-h treatment with 100 nM corticosterone caused 80 +/- 2% and 64 +/- 9% decreases in beta 1 levels in primary osteoblasts and ROS 17/2.8 cells, respectively. These results were confirmed with immunofluorescence microscopy, which showed a glucocorticoid-induced decrease in beta 1 staining. Treatment of primary rat osteoblasts and ROS 17/2.8 cells for 72 h with corticosterone also decreased beta 1-integrin messenger RNA levels in a dose-dependent manner. We have demonstrated that the inhibition of integrin expression by glucocorticoids is involved in the decrease in osteoblast adhesion to bone extracellular matrix proteins. These data suggest that integrin modulation may influence osteoblast function and bone formation and, thus, contribute to glucocorticoid-induced osteoporosis.

Estrogen prevents glucocorticoid-induced apoptosis in osteoblasts in vivo and in vitro.

The ability of estrogen to prevent glucocorticoid-induced apoptosis in osteoblasts was studied both in vitro and in vivo. Glucocorticoid treatment for 72 h produced a dose-dependent increase in the number of apoptotic cells, determined by acridine orange/ethidium bromide staining, with a maximal response of 31+/-2% and 26+/-3% with 100 nM corticosterone in primary rat and mouse osteoblasts, respectively. Simultaneous administration of varying concentrations of 17beta-estradiol and 100 nM corticosterone decreased apoptotic osteoblasts in a dose-dependent manner, with a maximal decrease of 70% with 0.01 nM 17beta-estradiol. Terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling also demonstrated glucocorticoid-induced DNA fragmentation that was inhibited by estrogen. Estrogen was shown to inhibit apoptosis induced by lipopolysaccharide treatment. As early as 6 h, Western blots demonstrated a dose-dependent decrease in the Bcl-2/Bax ratio, which reached a minimum of 0.18 in osteoblasts treated with 1000 nM corticosterone for 72 h. This reduction in Bcl-2/Bax was abolished by treating osteoblasts simultaneously with 17beta-estradiol, but not with 17alpha-estradiol. In 7-day-old mice, administration of varying concentrations of dexamethasone for 72 h resulted in a dose-dependent increase in the number of apoptotic osteoblasts as demonstrated by in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling staining of calvaria. A maximum of 22+/-1% apoptotic osteoblasts on the bone surface was found with 1 mg/kg BW dexamethasone compared with 2+/-1% in vehicle-treated mice. Injection of varying concentrations of 17beta-estradiol (0.5-5 mg/kg BW), but not 17alpha-estradiol, with 1 mg/kg dexamethasone produced a dose-dependent decrease in the number of apoptotic osteoblasts to 5+/-1% with 5 mg/kg 17beta-estradiol. Thus, glucocorticoid-induced apoptosis of osteoblasts may be prevented at least in part by 17beta-estradiol.

The interaction of heparin and basic fibroblast growth factor on collagen synthesis in 21-day fetal rat calvariae.

We examined the interactions of the glycosaminoglycan, heparin, and recombinant human basic fibroblast growth factor (bFGF) on collagen synthesis in 21-day fetal rat calvariae. In calvariae treated for 96 h, heparin (25 micrograms/ml) and bFGF (10(-9) M) inhibited collagenase-digestible protein (CDP) labeling by 52 and 60% of control, respectively, and the combination further inhibited CDP labeling. Inhibition of CDP labeling by heparin (25 micrograms/ml) or bFGF (10(-9), 10(-8) M) was similar in the presence or absence of aphidicolin (30 microM) an inhibitor of cell replication. Heparin selectively inhibited CDP labeling in the osteoblast rich central bone but bFGF alone or in combination with heparin inhibited CDP labeling both in the periosteum and central bone. Heparin and bFGF alone decreased steady state levels of alpha 1(I)procollagen messenger RNA (mRNA) at 24 h and the combination further decreased mRNA levels. A high concentration of insulin-like growth factor-1 (IGF-1, 3 x 10(-8) M) reversed the inhibitory effect of heparin on DNA synthesis and CDP labeling. In contrast, IGF-1 could not reverse the inhibitory effects of bFGF on CDP labeling but enhanced the stimulatory effects of bFGF on thymidine incorporation into DNA. We conclude that the inhibitory effects of heparin and bFGF on CDP are independent of effects on cell replication. We further conclude that both heparin and bFGF inhibit collagen synthesis at a pretranslational site since they decreased procollagen mRNA levels in osteoblasts. However, the inhibition of collagen synthesis by heparin and bFGF appears to involve divergent pathways since exogenous IGF-1 could overcome the effect of heparin but not bFGF on collagen synthesis.

Glucocorticoids inhibit fibronectin synthesis and messenger ribonucleic acid levels in cultured fetal rat parietal bones.

The effects of corticosterone on fibronectin production, bone growth, and morphology were examined in a mineralizing organ culture system derived from 20-day-old fetal rat parietal bones. During 4 days of culture, 1-1000 nM corticosterone had no significant effect on the increase in dry weight or on DNA content, but 100 and 1000 nM corticosterone did inhibit the increase in calcium content. Light microscopic examination of the 4-day cultures demonstrated a glucocorticoid-induced change in osteoblast shape and organization along the mineralizing front of the bone. A dose-dependent inhibition of fibronectin secretion into the medium was determined by enzyme-linked immunosorbent assay. In control cultures, fibronectin production was 0.105 +/- 0.005 microgram/ml.bone at 24 h and 0.397 +/- 0.037 microgram/ml.bone during the 72- to 96-h interval. The maximal inhibition of fibronectin secretion was 45% at 24 h and 70% at 96 h with 1000 nM corticosterone. Both immunofluorescent visualization of fibronectin staining in the tissue and a Western blot of fibronectin in the tissue showed a decrease in fibronectin levels. At 24 and 96 h, a dose- and time-dependent decrease in fibronectin mRNA transcripts was found. At 24 and 96 h, 1000 nM corticosterone produced a decrease of 42% and 62%, respectively, in fibronectin mRNA levels. Our findings show that glucocorticoids inhibit fibronectin production in developing bone. The decrease in fibronectin synthesis may contribute to altered osteoblast organization and function during bone formation.

Ascorbic acid alters collagen integrins in bone culture.

The effects of ascorbic acid on collagen synthesis, mineralization, and integrins were investigated in a mineralizing organ culture system derived from 20-day fetal rat parietal bones. A significant dose-dependent decrease in calcification at 96 h was demonstrated with decreasing concentrations of ascorbic acid (100-0 microg/ml). No effect on DNA content, [3H]thymidine incorporation, or dry weight was found in control (100 microg/ml ascorbic acid) bones compared with bones treated with decreased ascorbic acid concentrations (10, 1, and 0 microg/ml). Collagen synthesis, measured by [3H]proline incorporation, and alpha1(I) procollagen messenger RNA levels were also unaffected. However, ascorbic acid produced a dose-dependent decrease in the hydroxyproline content, with a maximal 76.8% decrease in bones without ascorbic acid compared with the control bones with 100 microg/ml ascorbic acid. Light microscopy of the ascorbic acid-deficient bones revealed a disruption of the osteoblast layer with misshapen osteoblasts and a decrease in the osteoid seam. The loss of osteoblast organization was also confirmed by analyzing the integrins for collagen by Northern and Western blot and immunofluorescence microscopy. A dose-dependent decrease in alpha2 and beta1 integrin messenger RNA levels and in alpha1, alpha2, and beta1 protein were found in 96-h bone cultures deficient in ascorbic acid. These integrin subunits mediate the binding of osteoblasts to collagen. Immunofluorescence microscopy also demonstrated a dose-dependent decrease in alpha2 and beta1 staining of the osteoblast layer. However, the protein levels of alpha3 and alpha5 subunits were not affected. No beta5 was detected, whereas only bones cultured without ascorbic acid demonstrated a small decrease in alpha(v) and beta3 protein levels. The alpha3, alpha5, alpha(v), and beta3 subunits are involved in cell binding to extracellular matrix proteins other than collagen. Thus, the integrins for collagen are down-regulated, probably in response to the underhydroxylated collagen fibrils, which causes a disruption of osteoblast organization leading to a decrease in mineralization of bone. Integrin assays for specific extracellular proteins may be useful tools in detecting matrix defects in various metabolic bone diseases.

Osteoblastic response to the defective matrix in the osteogenesis imperfecta murine (oim) mouse.

This work examines the cellular pathophysiology associated with the weakened bone matrix found in a murine model of osteogenesis imperfecta murine (oim). Histomorphometric analysis of oim/oim bone showed significantly diminished bone mass, and the osteoblast and osteoclast histomorphometric parameters were increased in the oim/oim mice, compared with wild-type (+/+) mice. To assess osteoblast activity, a rat Col1a1 promoter linked to the chloramphenicol acetyltransferase reporter transgene was bred into the oim model. At 8 d and 1 month of age, no difference in transgene activity between oim and control mice was observed. However, at 3 months of age, chloramphenicol acetyl transferase activity was elevated in oim/oim;Tg/Tg, compared with +/+;Tg/Tg and oim/+;Tg/Tg. High levels of urinary pyridinoline crosslinks in the oim/oim;Tg/Tg mice were present at all ages, reflecting continuing high bone resorption. Our data portray a state of ineffective osteogenesis in which the mutant mouse never accumulates a normal quantity of bone matrix. However, it is only after the completion of the rapid growth phase that the high activity of the oim/oim osteoblast can compensate for the high rate of bone resorption. This relationship between bone formation and resorption may explain why the severity of osteogenesis imperfecta decreases after puberty is completed. The ability to quantify high bone turnover and advantages of using a transgene that reflects osteoblast lineage activity make this a useful model for studying interventions designed to improve the bone strength in osteogenesis imperfecta.

Mice lacking the type I interleukin-1 receptor do not lose bone mass after ovariectomy.

We measured the effects of ovariectomy on the bone mass of mice that lacked type I interleukin-1 receptor (IL-I R1 -/- mice) in two genetic backgrounds (C57BL/6 x 129/Sv and C57BL/6) to investigate the role of interleukin-1 in the actions of estrogen on bone. At three weeks after surgery, ovariectomized wild-type mice decreased trabecular bone volume in the proximal humerus by 70% in a C57BL/6 x 129/Sv background and 48% in a C57BL/6 background compared to sham-operated controls. In contrast, there was no significant decrease in trabecular bone mass in IL-1 R1 -/- mice after ovariectomy. The estrogen status of all groups was confirmed by measurement of uterine wet weight. These results demonstrate that a functional IL-1 response pathway is required for mice to lose trabecular bone mass after ovariectomy in this model and they imply that IL-1 is an important mediator of the effects of ovariectomy on bone mass. Hence, therapeutic interventions that block the effects of IL-1 on bone may be beneficial for treating diseases of rapid bone loss such as post-menopausal osteoporosis.