Citations

Abstract The osteoblast plays a critical role in bone formation, bone remodeling, bone matrix formation, and matrix calcification. To better understand the process of osteoblast-controlled bone formation, we determined the structure and isoform types of the plasma membrane calcium pump from normal human osteoblasts. A complementary DNA library from normal human osteoblasts was screened for plasma membrane calcium pump clones. Sequencing and analysis of cDNA clones revealed the presence of a 3986 base pair cDNA that encoded a 1220 amino acid protein that was similar to the human plasma membrane calcium pump isoform 1. Polyadenylated RNA from human osteoblast cells contains bands of RNA approximately 5050 and 6750 bases long. Reverse transcription of polyadenylated RNA from human osteoblasts followed by amplification of the RNA-DNA duplex with calcium pump isoform-specific primers revealed the presence of isoforms 1 and 2 of the calcium pump. Isoform 4 was not detected. We conclude that normal adult human osteoblasts contain a plasma membrane calcium pump that is similar to the human plasma membrane calcium pump isoform 1. It is likely that this pump plays an important role in the cell biology of the human osteoblast.

Abstract We assessed the cross-sectional relationship of age, menopausal years, body mass, previous estrogen use, and ethnic background to bone mineral status in a sample of 875 healthy postmenopausal women at the time they were recruited from the community to participate in a multicenter clinical trial. The women were 1-10 years postmenopause, 45-64 years of age, and had not received estrogen replacement therapy within 3 months of enrollment. Of the participants, 89% were white, 69% had a spontaneous menopause, and 53% had a history of previous estrogen replacement therapy. Bone mineral density (BMD) of the lumbar spine (L2-4) and proximal femur was measured by dual-energy x-ray absorptiometry. Results were consistent with a significant negative linear regression of BMD on age or years from menopause. Body mass index (BMI) correlated significantly with BMD at all sites (L 2-4 r = 0.28; femoral neck r = 0.34, p < 0.0001). BMD adjusted for age and BMI were higher at both sites in women who had taken estrogen versus those who had not (L2-4 0.976 + 0.009 versus 0.932 + 0.01; femoral neck 0.740 + 0.006 versus 0.708 + 0.008, p < 0.05). Adjusted BMD also increased with duration of ERT. Parity was negatively associated with L2-4 BMD (p = 0.03) but did not correlate significantly with BMD at the femoral neck. Black women had the highest L2-4 BMD, and Hispanic women had the highest femoral neck BMD, even when results were adjusted for age and BMI. When data were corrected for differences in bone size, these interethnic differences were no longer significant. We conclude that increased body mass is positively correlated with BMD, and this may confer a degree of skeletal protection to heavier postmenopausal women. Exposure for 5 years to exogenous estrogen is associated with significantly increased age- and BMI-adjusted BMD.

Abstract Calcein was given to growing male rats to label mineralizing bone, and groups of animals were sacrificed from 6 h to 12 days later. Fluorochrome-labeled perimeter and osteoblast perimeter were determined in a growth-adjusted sampling site in the secondary spongiosa of the proximal tibial metaphysis. The best correspondence between calcein-labeled perimeter and osteoblast-lined perimeter was during the initial 24 h following administration of the label. This association decreased progressively thereafter because of (1) cessation of bone formation on fluorochrome-labeled surfaces and (2) initiation of new foci of bone formation. The time-dependent decline in osteoblast-lined trabeculae with adjacent calcein label exceeded the increase in osteoblast perimeter on nonfluorochrome-labeled trabeculae, indicating that there was a net decrease in osteoblast number. Fluorochrome-labeled perimeter and bone area showed parallel decreases with time after labeling, as a result of bone resorption. Interestingly, the decrease in cancellous bone was caused exclusively by a reduction in trabecular number. There was no change in trabecular thickness. The findings suggest that the decreased osteoblast number and progressive bone resorption resulted in complete erosion of trabeculae most distal to the growth plate. As a result of the nearly equal growth and destruction of trabeculae, there was no change in the length of the cancellous metaphysis with time. It is concluded that cancellous bone turnover in growing rats represents a maturation process that differs fundamentally from bone turnover in adults.

Abstract Interleukin 1αa (IL-1α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) are cytokines with potent bone-resorbing effects; some of these biologic effects are opposed by interleukin-1 receptor antagonist (IL-1ra). In vitro and animal model studies suggest that these cytokines are paracrine mediators of the increased bone resorption associated with estrogen deficiency, and increases in their production also could contribute to age-related bone loss. Therefore, we measured serum concentrations of these cytokines in 80 normal women who were 24-87 years old. IL-6 concentration correlated highly with age (p > 0.001) and increased three-fold during life. However, multiple-regression analysis showed no significant correlation between serum IL-6 levels and menopausal status, serum estradiol concentration, or markers for bone turnover (serum bone alkaline phosphatase, osteocalcin, carboxyl-terminal telopeptide of type I collagen, or 24 h urinary free pyridinoline excretion). Serum IL-1α, IL-1β, or IL-1ra level did not change with age and, by multiple-regression analysis, did not correlate with markers of bone turnover, except IL-1ra weakly with ICTP. We found no relationship between bone-resorbing cytokines and ovarian function. Although the large age-related increase in serum IL-6 concentration could contribute to age-related bone loss, the lack of correlation with markers for bone turnover argues against this. However, based on the strong evidence in experimental animals that these cytokines are involved in estrogen action on bone, further studies in humans are warranted.

Abstract Cell-cell interactions are important in the regulation of endocrine cell secretion. To investigate the possibility that cell communication may alter the regulation of parathyroid cell secretion, we utilized the reverse hemolytic plaque assay (RHPA) to measure parathyroid hormone (PTH) release from individual cells. Bovine parathyroid cells were dispersed and plated with protein A-conjugated erythrocytes at cell densities ranging from 0.9 to 36 X 102 cells/cm2 in 0.2 mM calcium. Cell populations were greater than 98% homogeneous as determined by immunocytochemistry and in situ hybridization for PTH mRNA. Plaques were developed and data analyzed for the amount of PTH per cell released (plaque area in μm2 X 104) and the determination of cell recruitment (% plaques formed). A positive correlation existed between parathyroid cell density and the amount of PTH released. As the distance between cells increased, the plaque area (amount of PTH released per cell) decreased (ranging from 1.0 X 1044 μm2 at 0.9 X 102 cells/cm2 versus 1.6 X 104 μm2 at 36 X 102 cells/cm2). The percentage of cells releasing PTH (recruitment) also decreased (16% at 0.9 X 102 cells/cm2 versus 47% at 36 X 102 cells/cm2). These data suggest that parathyroid cells in close proximity are stimulated to secrete more hormone than those at lesser densities. In addition, parathyroid cells are recruited to secrete PTH when plated at high density. Factor(s) released by the parathyroid cell may increase cell responsiveness and stimulate secretion in a paracrine fashion.

Abstract A quality control (QC) program for bone mineral measurements at the proximal femur by dual-energy x-ray absorptiometry (DXA) was designed for the osteoporosis component of the Third National Health and Nutrition Examination Survey (NHANES III). Major elements of the QC program are (1) a QC center for review of all scans, (2) setup procedures for and continuous monitoring of daily QC procedures, (3) reference standards for cross-calibration, (4) longitudinal studies for assessment of instrument stability, (5) monitoring of technologist performance, and (6) training. This report describes the results of the QC program of the first half of this 6 year study, which began in 1988. Measurements were performed on 7376 subjects in three mobile examination centers, which traveled to a new location about every 3 months, a total of 44 locations. A small percentage (3.5%) of all scans were rejected, mostly because of patient motion during scanning, but 33% of the remaining scans required reanalysis at the QC center to refine the location of the regions of interest. Precision in spine and hip phantoms was below 1% at all ROIs. In 535 subjects with duplicate scans, age 20–91 of both sexes, examined in a blinded review, precision for BMD at the femur neck ROI was 3.2% (CV) and 5.1% for Ward's triangle BMD. The central review improved scan quality, increased the number of usable scans, and reduced significantly the range of the mean percentage difference in the duplicate scans. Minor alterations in machine function were observed and corrected. QC results on phantoms were similar to those obtained with stationary instruments in dedicated laboratories. Retrospective analysis of the regression slopes from QC records did not show a need for correction of the data base, indicating that the instruments were stable during the course of the study.

Abstract Mutations in one of the two genes encoding type I procollagen (COL1A1 and COL1A2) are frequently the cause of osteogenesis imperfecta (OI), a disorder characterized by brittle bones. Here we tested whether patients with low bone density also have mutations in these genes. The 26 patients studied had no apparent metabolic bone disease, but most had a positive family history of osteopenia or osteoporosis. Although a diagnosis of OI was considered by the clinician in some cases, the clinical criteria for OI were not satisfied. Our strategy for mutation analysis consisted of PCR amplification of cDNA made to fibroblast mRNA using primers specific for the coding regions of COL1A1 and COL1A2. The PCR products were then sequenced directly with primers located within each PCR product. We found that 3 of 26 patients had mutations that altered the encoded amino acid. One mutation, at position α2(I)-661 has been reported (Spotila et al. 1991 Proc Natl Acad Sci USA PNAS 88: 5423). The other 2 patients, who were not related to each other, had a mutation that altered the proline codon at α1(I)-27 to alanine. This mutation was not found in 81 normal individuals or in 37 additional osteopenic individuals. However, its effect on the biologic function of type I collagen, as well as its role in osteopenia, is uncertain. In addition to the two mutations, we found a polymorphism in codon α2(I)-459. Although this polymorphism involved an amino acid substitution, it was present with equal frequency in the patient and the normal population. By analyzing this and previously reported neutral sequence variants in the COL1A2 gene, we determined that all patients expressed both alleles of the COL1A2 gene. The 12 patients who were heterozygous for a COL1A1 neutral sequence variant also expressed both alleles. Here we present all PCR primer and sequencing primer information. The results suggest that surveying a larger group of similarly selected individuals may reveal additional mutations in the COL1A1 or COL1A2 genes.

AbstractIn a population-based retrospective cohort study, Rochester women aged 35–69 years who were first diagnosed with one or more vertebral fractures in 1950–1979 were followed for the development of a subsequent hip fracture. The 336 women with no history of hip fracture at the time of their vertebral fracture experienced 52 proximal femur fractures in 4788 person-years of follow-up. The standardized morbidity ratio (SMR) of observed to expected hip fractures was 1.8 (95% CI, 1.3–2.4) and was higher for intertrochanteric than cervical femoral fractures (SMR, 2.3 versus 1.3; P = 0.07). Hip fracture risk among women with symptomatic vertebral fractures was slightly less than in those with asymptomatic vertebral fractures (SMR, 1.8 versus 2.3; not significant), and younger women had no higher risk of a subsequent hip fracture than women who were ≥60 years of age at the time of their vertebral fracture (SMR, 1.4 versus 1.8; not significant). Alternative explanations are possible, but these data are consistent with heterogeneity in the pathogenesis of different osteoporotic fractures.

Abstract The coupling of bone resorption and formation suggests that autocrine and paracrine factors are produced and released within the local bone environment. Since osteoblasts secrete latent transforming growth factor β (TGF-β), and osteoclasts can activate this latent complex, one candidate paracrine coupling factor is TGF-β. To examine whether TGF-β is also a potential osteoclast-derived autocrine and paracrine factor, highly purified avian osteoclasts were examined for synthesis of TGF-β and the mechanism by which osteoclasts activate the latent TGF-β complex was investigated. TGF-β protein production was measured within 4 h of culture in osteoclast-conditioned media using growth factor inhibition of CCL-64 cells and verified by blocking effects with anti-TGF-β antibodies. Synthesis of TGF-β was confirmed by northern blotting and metabolic labeling. Northern blots of total RNA revealed that osteoclasts expressed the mRNA for TGF-β2, β3, and β4. Biosynthetic studies suggest that TGF-β2 was the principle form secreted in culture. Nearly all the TGF-β that was secreted had been activated by the osteoclasts. When presented with exogenous latent TGF-β, osteoclasts activated latent TGF-β from a variety of sources. Furthermore, osteoclast-conditioned media retained the ability to activate latent TGF-β during cell-free incubations at 37°C. Osteoclast-mediated activation was inhibited by proteinase inhibitors, weak base treatment of the cells, or disruption of the cytoskeletal network. These data suggest that osteoclasts may secrete proteinases into the extracellular milieu to activate latent TGF-β and that TGF-β may be an autocrine factor involved in regulating osteoclast activity.

Abstract In a 4 year clinical trial in 202 postmenopausal osteoporotic women receiving NaF at 75 mg/day or placebo (both groups received supplementary calcium at 1500 mg/day), we found (N Engl J Med 322:801, 1990) that NaF increased bone mineral density in the lumbar spine (LS-BMD) substantially but did not decrease vertebral fracture rate (VFR), and it increased the nonvertebral fracture rate. Additional analyses and extended observations are now available on 50 women from the NaF group followed for up to 6 years of treatment. In these women, LS-BMD increased linearly over the 6 years (median rate, 8.7%/year or 0.063 g/cm2/year). Because during the 4 year trial the NaF dosage was decreased (because of side effects) in 54 of the 101 women randomized to NaF, dose-response relationships could be evaluated. For the entire study population, serum F level correlated directly with increase in LS-BMD (r = 0.61, P < 0.001). When individual person-years of observation were grouped by deciles of LS-BMD, VFR (per 100 person-years) decreased to a nadir of 24 as mean LS-BMD for the group increased from 0.6 to 1.2 g/cm2 and then doubled to 52 in the group with mean LS-BMD of 1.6 g/cm2. Multivariate analyses and inspection of three-dimensional plots revealed a complex pattern in which VFR was influenced by interaction of several variables. When the effects of LS-BMD, changes in LS-BMD, and serum F were assessed simultaneously, VFR was seen to decrease with increasing LS-BMD except when the higher LS-BMD was associated with rapid rate of increase in LS-BMD or a large increase from baseline serum F. For some patients (noncompliers or nonresponders), serum F or LS-BMD failed to increase. Thus, it is possible that lower dosages of NaF produce moderate decreases in VFR.