Citations

Abstract To determine if bone cells produce interleukin-1β (IL-1β), a potent bone resorption-stimulating agent, we studied well-characterized, nearly homogeneous cultures of normal human osteoblast-like (hOB) cells. With four strains of such cells, vehicle-treated cultures produced minimal IL-1β (mean ± SEM, 1.3 ± 0.3 pg/ml per 106 cells per 24 h) and showed dose-dependent (r = 0.99) increases to 2.2 ± 0.7, 5.0 ± 0.9, or 17.8 ± 6.7 pg/ml, respectively, after treatment with lipopolysaccharide (LPS) at 3, 10, or 30 μg/ml (for increases after 10 and 30 μg/ml treatments, P < 0.05). After treatment with tumor necrosis factor α (TNF-α at 10 U/ ml, IL-1β increased to 16.2 * 3.7 pg/ml (P < 0.05). Neither 17β-estradiol nor bovine parathyroid hormone(1–34) (each at 10 nM), alone or in combination with LPS or TNF-α, affected IL-1β release. Northern blot analysis of total cellular RNA preparation revealed a single hybridization band at 1.9 kb when probed with a partially deleted cDNA for human IL-1β. The steady-state IL-1β mRNA levels showed a significant increase with LPS treatment and a lesser increase with TNF-α treatment in hOB cells. Moreover, TNF-α produced an even greater increase in IL-1 mRNA in HOBIT cells, a well-differentiated clonal cell line derived from normal hOB cells transfected with the SV40 large T antigen. We conclude that human cells of the osteoblast lineage produce IL-Iβ in response to well-recognized stimuli for IL-1 release from responsive tissue. Therefore, IL-1 may play an important role in the local regulation of bone remodeling.

Abstract The NH2-terminal cleavage peptide of procalcitonin (N-proCT) recently was reported to be a bone cell mitogen (Burns DM et al., Proc Natl Acad Sci USA 86:9519–9523, 1989). We have investigated the effect of N-proCT on the proliferation of normal human cells that have the phenotype of mature osteoblasts (hOB cells). N-proCT treatment for 24, 48, or 96 h in concentrations from 1 nM to 1 μM did not significantly increase [3H]thymidine uptake (means ranged from -19% to 38% of control, no significant differences) in hOB cells (6–10 cell strains per experiment) plated at four different densities. However, the hOB cells responded significantly to treatment with transforming growth factor β (3 ng/ml), bovine insulin (300 μg/ml), or 30% fetal calf serum, which were included in all experiments as positive controls. The [3H]thymidine uptake data were confirmed in a direct cell count experiment tested at 96 h. Thus our data do not support the hypothesis that N-proCT is a potent mitogen for normal human osteoblasts.

Abstract Although osteoblasts contain estrogen receptors, it is unclear whether estrogen has direct effects on osteoblast proliferation and differentiation. We evaluated the effects of 17β-estradiol treatment (1 pM to 10 nM) on the proliferation and differentiation of cultured normal adult human cells that expressed many of the phenotypic characteristics and hormonal sensitivities of mature osteoblasts (hOB cells). Treatment of hOB cells with estradiol for as long as 144 h did not affect the rate of DNA synthesis and had minimal, if any, effects on differentiated function. Whereas alkaline phosphatase activity was increased by nearly twofold (P < 0.01) when the hOB cells were treated with 1 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], treatment with estradiol had no effect when given alone and did not affect the cells' response to 1,25-(OH)2D3. Similarly, the release of bone gla protein (BGP, osteocalcin) was induced by treatment with 1,25-(OH)2D3 (P < 0.05), but estradiol treatment did not affect this response. Cellular levels of mRNA for alkaline phosphatase and BGP were not altered by estradiol treatment. We conclude that estradiol treatment does not have major effects on the growth or differentiation of cultured hOB cells. These results are consistent with previous observations in vivo that indicate estrogen acts principally to decrease bone resorption, not to modulate its formation.

Abstract Although it is a cardinal feature of involutional osteoporosis, there is often disagreement on what constitutes a vertebral fracture. We measured vertebrae T4-L5 in 52 healthy women to develop a normal range (mean ± 3 SD) for vertebral shape and used these data to assess the prevalence of vertebral fractures. We classified vertebral fractures by type of deformity (wedge, biconcavity, or compression) and further by the degree of deformity (grades 1 and 2). In 195 postmenopausal women who were an age-stratified random sample of the Rochester population (ages 47–94), 40 (21%) had vertebral fractures (mean, 2 per person). There was a similar number of compression and wedge fractures, and grade 2 fractures were as common as grade 1. In a referral sample of 74 women with suspected osteoporosis, 62 (84%) had vertebral fractures (mean, 3.3 per person). Wedge fractures were most common, and grade 2 fractures were more common than grade 1. The distribution of type and grade of fractures differed between the two patient groups (P < 0.01). Bone mineral density of the lumbar spine was related to mean fracture grade (r = −0.33, P < 0.05) and to fracture number (r = −0.57, P < 0.001) but not to fracture type. We conclude that a comprehensive approach is required in describing vertebral fractures. Using this approach we found distortion in the fracture characteristics of women referred to an osteoporosis clinic compared to women in the community.

Abstract Osteoporosis is widely viewed as a major public health concern, but the exact magnitude of the problem is uncertain and likely to depend on how the condition is defined. Noninvasive bone mineral measurements can be used to define a state of heightened fracture risk (osteopenia), or the ultimate clinical manifestation of fracture can be assessed (established osteoporosis). If bone mineral measurements more than 2 standard deviations below the mean of young normal women represent osteopenia, then 45% of white women aged 50 years and over have the condition at one or more sites in the hip, spine, or forearm on the basis of population-based data from Rochester, Minnesota. A smaller proportion is affected at each specific skeletal site: 32% have bone mineral values this low in the lumbar spine, 29% in either of two regions in the proximal femur, and 26% in the midradius. Although this overall estimate is substantial, some other serious chronic diseases are almost as common. More importantly, low bone mass is associated with adverse health outcomes, especially fractures. The lifetime risk of any fracture of the hip, spine, or distal forearm is almost 40% in white women and 13% in white men from age 50 years onward. If the enormous costs associated with these fractures are to be reduced, increased attention must be given to the design and implementation of control programs directed at this major health problem.

Abstract High caffeine consumption has been proposed as a risk factor for osteoporotic fracture, but the evidence associating high caffeine intake with low bone density is inconsistent. We therefore examined the influence of caffeine consumption on bone mineral at six skeletal sites in an age-stratified random sample of white women residing in Rochester, Minnesota. After age adjustment, there was no association between overall caffeine consumption and bone mineral at five of the six sites. In the femoral shaft, however, there was a statistically significant interaction between age and caffeine consumption so that high caffeine intake was associated with slight reductions in bone mineral among elderly subjects but with modestly increased bone mineral at younger ages. When caffeine intake was categorized by source, no consistent influence of coffee, tea, or other caffeinated beverage consumption could be detected on bone mineral. Caffeine intake was, however, positively associated with cigarette smoking and alcohol consumption. After adjusting for age, caffeine consumption was not correlated with biochemical indices of bone turnover, circulating concentrations of estradiol and estrone, or other dietary and musculoskeletal variables. These data suggest that caffeine intake in the range consumed by a representative sample of white women is not an important risk factor for osteoporosis. Among elderly women, however, in whom calcium balance performance is impaired, high caffeine intake may predispose to cortical bone loss from the proximal femur.

Abstract A new bone cell line was established by transfecting normal adult human osteoblast-like (hOB) cells, derived from a 68-year-old woman, with the plasmid pSV3 neo. The plasmid included coding sequences and promotors for the large and small T antigens of the SV40 virus as well as resistance to the antibiotics neomycin and G418. A single antibiotic-resistant colony was located and cloned. Large tumor antigen production in the clonal cell line was confirmed by indirect immunofluorescence study. Treatment with 1,25-dihydroxy-vitamin D3 increased steady-state concentrations of protein and mRNA for osteocalcin and for alkaline phosphatase. Northern blot analyses also demonstrated the presence of mRNAs for α(I)-procollagen, osteopontin 1a, transforming growth factor β, and interleukin-1β. The plasma membrane calcium pump and osteonectin were identified by immunocytochemical analysis. These cells produced a matrix that mineralized when β-glycerophosphate was added to their cultures. As assessed by functional receptor assays, both estrogen and androgen receptors were present and functional, although at low concentrations. Treatment with parathyroid hormone did not stimulate adenylate cyclase activity. Thus, these cells are a well-differentiated, steroid-responsive clonal cell line that closely approximates the phenotype of the mature osteoblast. They should serve as an excellent model for the study of osteoblast biology.

Abstract Silicon in trace amounts enhances bone formation, and the silicon-containing compound zeolite A (ZA) increases eggshell thickness in hens. In the studies reported here, treatment of nearly homogeneous strains of normal human osteoblast-like cells for 48 h with ZA at 0.1–100 μg/ml induced a dose-dependent increase (r = 0.35, P < 0.001) in DNA synthesis (n = 31) to 162 ± 16% (mean ± SEM) of control and in the proportion of cells in mitosis (n = 4) from 9.1 ± 1.8 to 27.0 ± 4.5% (r = 0.69, P < 0.005). ZA treatment also increased alkaline phosphatase activity (P < 0.05) and osteocalcin release (P < 0.05) but did not significantly affect collagen production per individual cell. The mitogenic action of ZA was dependent on cell seeding density over the range of 1250–40,000 cells per cm2, which is consistent with induction of an autocrine factors). TGF-β is a potent mitogen for osteoblasts. ZA treatment increased the steady-state mRNA levels of transforming growth factor β1 (TGF-β1) and induced the release of the latent form of TGF-β protein into the conditioned medium within 6 h. We conclude that ZA induces the proliferation and differentiation of cells of the osteoblast lineage.