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Clinical diagnosis of osteoporosis is generally based on the presence of a symptomatic fracture because this provides the clearest definitions of the disease. In some cases osteoporosis is iatrogenic but with the exception of steroid-induced osteoporosis this is a rare finding. Hormonal inbalance may result in osteoporosis, with the disease usually being clearly related to an excessive level of the hormone in the serum. Preventive treatment with either hormones or calcium supplements may be useful if initiated before the mass of bone has decreased to a level at which minor trauma will cause fracture. Fluoride has been used in the past for the treatment of osteoporosis or bone losing diseases such as multiple myeloma and hypercortisonism. The mechanism of action of fluoride on the mineralized tissue, in this instance bone, is also different from those in the prevention of dental caries: It is one of osteoblastic stimulation, rather than the prevention of crystal dissolution.

Using combined dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography, we demonstrate that men matched with women for femoral neck (FN) areal bone mineral density (aBMD) have lower volumetric BMD (vBMD), higher bone cross-sectional area, and relatively similar values for finite element (FE)-derived bone strength. aBMD by DXA is widely used to identify patients at risk for osteoporotic fractures. aBMD is influenced by bone size (i.e., matched for vBMD, larger bones have higher aBMD), and increasing evidence indicates that absolute aBMD predicts a similar risk of fracture in men and women. Thus, we sought to define the relationships between FN aBMD (assessed by DXA) and vBMD, bone size, and FE-derived femoral strength obtained from quantitative computed tomography scans in men versus women. We studied men and women aged 40 to 90 years and not on osteoporosis medications. In 114 men and 114 women matched for FN aBMD, FN total cross-sectional area was 38% higher (P

Bone strength at the ultradistal radius, quantified by micro-finite element modeling, can be predicted by variables obtained from high-resolution peripheral quantitative computed tomography scans. The specific formula for this bone strength surrogate (-555.2 + 8.1 × [trabecular vBMD] + 19.6 × [cortical area] + 4.2 × [total cross-sectional area]) should be validated and tested in fracture risk assessment.The purpose of this study was to identify key determinants of ultradistal radius (UDR) strength and evaluate their relationships with age, sex steroid levels, and measures of habitual skeletal loading.UDR failure load (~strength) was assessed by micro-finite element (μFE) modeling in 105 postmenopausal controls from an earlier forearm fracture case-control study. Predictors of bone strength obtained by high-resolution peripheral quantitative computed tomography (HRpQCT) in this group were then evaluated in a population-based cohort of 214 postmenopausal women. Sex steroids were measured by mass spectrometry.A surrogate variable (-555.2 + 8.1 × [trabecular vBMD] + 19.6 × [cortical area] + 4.2 × [total cross-sectional area]) predicted UDR strength modeled by μFE (R(2) = 0.81), and all parameters except total cross-sectional area declined with age. Evaluated cross-sectionally, the 21% fall in predicted bone strength between ages 40-49 years and 80+ years more resembled the change in trabecular volumetric bone mineral density (vBMD) (-15%) than that in cortical area (-41%). In multivariable analyses, measures of body composition and physical activity were stronger predictors of UDR trabecular vBMD, cortical area, total cross-sectional area, and predicted bone strength than were sex steroid levels, but bio-available estradiol and testosterone were correlated with body mass.Bone strength at the UDR, as quantified by μFE, can be predicted from variables obtained by HRpQCT. Predicted bone strength declines with age with changes in UDR trabecular vBMD and cortical area, related in turn to reduced skeletal loading and sex steroid levels. The predicted bone strength formula should be validated and tested in fracture risk assessment.

Compared to white women, lower areal bone mineral density (aBMD) in middle-aged Vietnamese immigrants is due to reduced trabecular volumetric bone mineral density (vBMD), which in turn is associated with greater trabecular separation along with lower estrogen levels.The epidemiology of osteoporosis in Asian populations is still poorly known, but we previously found a deficit in lumbar spine aBMD among postmenopausal Southeast Asian women, compared to white women, that persisted after correction for bone size. This issue was revisited using more sophisticated imaging techniques.Twenty Vietnamese immigrants (age, 44-79 years) were compared to 162 same-aged white women with respect to aBMD at the hip, spine and wrist, vBMD at the hip and spine by quantitative computed tomography and vBMD and bone microstructure at the ultradistal radius by high-resolution pQCT. Bone turnover and sex steroid levels were assessed in a subset (20 Vietnamese and 40 white women).The aBMD was lower at all sites among the Vietnamese women, but femoral neck vBMD did not differ from middle-aged white women. Significant differences in lumbar spine and ultradistal radius vBMD in the Vietnamese immigrants were due to lower trabecular vBMD, which was associated with increased trabecular separation. Bone resorption was elevated and bone formation depressed among the Vietnamese immigrants, although trends were not statistically significant. Serum estradiol was positively associated with trabecular vBMD in the Vietnamese women, but their estrogen levels were dramatically lower compared to white women.Although reported discrepancies in aBMD among Asian women are mainly an artifact of smaller bone size, we identified a specific deficit in the trabecular bone among a sample of Vietnamese immigrants that may be related to low estrogen levels and which needs further study.

The concept that vertebral fractures are caused by excessive loss of cancellous bone has been challenged by a recent study (J Bone Min Res 2:221, 1987) suggesting that vertebral bodies are composed mainly of cortical bone rather than cancellous bone. To resolve disagreement we used two independent methods to quantify the proportions of cortical and cancellous bone in 400 microns thick sections of the bodies of the second lumbar vertebrae from six men (aged 21-58 years) and seven women (aged 25-58 years). Based on the ash weight of the manually dissected components, 80% of the total bone in men and 72% in women was cancellous bone. Based on computer-assisted scanning of sections with a microdensitometer, 81% of the total bone in men and 71% in women was cancellous bone. We conclude that the traditional concept is correct: the vertebral body is composed mainly of cancellous bone.

We report the establishment of a human fetal osteoblast cell line derived from biopsies obtained from a spontaneous miscarriage. Primary cultures isolated from fetal tissue were transfected with a gene coding for a temperature-sensitive mutant (tsA58) of SV40 large T antigen along with a gene coding for neomycin (G418) resistance. Individual neomycin resistant colonies were screened for alkaline phosphatase (AP)-specific staining. The clone with the highest AP level, hFOB 1.19, was examined further for other osteoblast phenotypic markers. Incubation of hFOB cells at the permissive temperature (33.5 degrees C) resulted in rapid cell division, whereas little or no cell division occurred at the restrictive temperature (39.5 degrees C). Both AP activity and osteocalcin (OC) secretion increased in a dose-dependent manner following dihydroxyvitamin D3 (1,25-D3) treatment when cultured at either temperature. However, AP and 1,25-D3-induced OC levels were elevated in confluent hFOB cells cultured at 39.5 degrees C compared with 33.5 degrees C. Treatment of hFOB cells with 1-34 parathyroid hormone (PTH) resulted in an increase in cAMP levels. Upon reaching confluence, hFOB cultures went through programmed differentiation and formed mineralized nodules as observed by von Kossa staining. Further, immunostaining of postconfluent, differentiated hFOB cells showed that high levels of osteopontin, osteonectin, bone sialoprotein, and type I collagen were expressed. Therefore, the clonal cell line hFOB 1.19 provides a homogeneous, rapidly proliferating model system to study certain stages of human osteoblast differentiation.

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.

In bone marrow aspirates from postmenopausal women, systemic estrogen treatment decreased differentiation of mononuclear progenitor cells toward a more mature osteoclast phenotype. This was not associated with changes in surface receptor for proresorptive cytokines.Although mechanisms by which estrogen (E) decreases bone resorption have been extensively studied in rodents, little information is available in humans.In bone marrow aspirates from 34 early postmenopausal women randomly assigned to receive 4 weeks of treatment (100 microg/day of transdermal 17beta-estradiol) or no treatment, we assessed osteoclast differentiation and surface receptors using flow cytometry with fluorescent-labeled specific antibodies.E treatment decreased (P0.05) the proportion of bone marrow mononuclear cells (BMMNCs) expressing the calcitonin receptor (CTR), a late osteoclast phenotype marker. There was an increase in c-Fms concentration in osteoclast lineage cells (P0.05) and in the proportion of BMMNCs expressing TNFR2 (P0.05), but there were no significant effects on other surface receptors for proresorptive factors (RANK, TNFR1, TREM2, or OSCAR). Changes in serum CTx and TRAP 5b, markers for bone resorption, correlated directly (P0.05) with the proportion of BMMNCs expressing CTR and, for TRAP 5b only, TNFR2 and inversely with c-Fms concentration (all P0.05).E reduces bone resorption, in part, by decreasing differentiation of BMMNCs into mature osteoclasts. This action cannot be explained by decreased concentrations of surface receptors for proresorptive factors. The roles of increases in c-Fms concentration and the proportion of TNFR2((+)) cells are unclear.

Approaches for recognizing vertebral fractures remain controversial.An age-stratified population sample of 512 postmenopausal women was followed with serial radiographs for up to 12 years (4455 person-years).112 women experienced a new vertebral fracture (20% reduction in any vertebral height from baseline) within this study period, for an annual age-adjusted (to US white womenor =50 years of age in 2000) incidence of 23 per 1000. Depending on the morphometric definition used, the prevalence of vertebral deformities at baseline ranged from 3 to 90%. A recent method to standardize vertebral heights produced the best agreement with a qualitative clinical reading of the films [kappa (kappa), 0.53]. Almost all of the different baseline definitions predicted future vertebral fractures, but most of the predictive power was attributable to the severe (e.g., 4 SD) deformities included within more generous (e.g., 3 SD) classifications. Whereas the generous definitions were more sensitive, and the restrictive ones more specific, their overall abilities to predict a new vertebral fracture were roughly comparable as evaluated by the c-index (analogous to the area under an ROC curve).This result suggests that the choice of a morphometry definition depends on the particular application and, in particular, on whether it is more important to maximize sensitivity or specificity.

Genistein, a soybean isoflavone, has estrogen-like activity in mammals, including the prevention of bone loss. However, whether its mechanism of action on bone turnover is distinct from that of estrogen or raloxifene is unknown. Although genistein has been reported to bind both estrogen receptor (ER) isoforms (alpha and beta), little is known concerning differential activation of gene expression via these ER isoforms. To examine this question, comparison of the responses of normal fetal osteoblast (hFOB) cells stably expressing either ERalpha (hFOB/ERalpha9) or ERbeta (hFOB/ERbeta6), to treatment with genistein, 17beta-estradiol (E(2)) or raloxifene were conducted. In hFOB/ERalpha9 cells, both genistein and E(2) increased the endogenous gene expression of the progesterone receptor (PR), the proteoglycan versican, and alkaline phosphatase (AP), but inhibited osteopontin (OP) gene expression and interleukin-6 (IL-6) protein levels. Raloxifene had no effect on these bone markers. Genistein, but not raloxifene, also mimicked E(2) action in the hFOB/ERbeta6 cells increasing PR gene expression and inhibiting IL-6 production. To determine whether the gene regulatory actions of genistein in human osteoblast cells occur at the level of transcription, its action on the transcriptional activity of a PR-A promoter-reporter construct was assessed. Both genistein and E(2) were found to stimulate the PR promoter in the hFOB cell line when transiently co-transfected with either ERalpha or ERbeta. Whereas hFOB cell proliferation was unaffected by E(2), raloxifene or genistein at low concentrations, higher concentrations of genistein, displayed significant inhibition. Together, these findings demonstrate that genistein behaves as a weak E(2) agonist in osteoblasts and can utilize both ERalpha and ERbeta.

Estrogen is a major sex steroid that affects the growth, maintenance, and homeostasis of the skeleton. Two isoforms of the estrogen receptor (ERalpha and ERbeta) mediate the transcriptional effects of estrogen. Although both isoforms of ER are present and functional in some human osteoblast (OB) cell lines, there is minimal information on the differential regulation of transcription by ERalpha and ERbeta homo- or heterodimers. This report demonstrates that ERalpha and ERbeta coexpression decreases the transcriptional capacity (relative to each ER isoform alone) on an estrogen response element-dependent reporter gene in OBs but not in other non-osteoblastic cell lines. These data suggest that ERalpha and ERbeta coexpression can differentially influence the degree of transcriptional activation in certain cell types. Interestingly, the overexpression of the steroid hormone receptor coactivator-1 (SRC1) resulted in preferential transcriptional enhancement by ERbeta as well as coexpressed ERalpha and ERbeta, whereas SRC2 overexpression appeared to preferentially enhance ERalpha transactivation. SRC3 overexpression failed to enhance estrogen-dependent transcription of any ER combination in OBs. Similar overexpression experiments in COS7 cells exhibited preferential enhancement of ERalpha function with all SRCs, including SRC3. Our data also demonstrated that SRC3 mRNA is reduced in osteoblastic cells, suggesting that SRC3 may have only a minor role in these cells. These data suggest that the transactivation capacity of various ER isoforms is both SRC species and cell type dependent.

The epidemiology of bone loss in populations of African heritage is still poorly known. We compared a convenience sample of 47 African-American (AA) residents of Rochester, Minnesota (32 women, 15 men) and 66 recent immigrants from Somalia (all women) with 684 white subjects (349 women, 335 men) previously recruited from an age-stratified random sample of community residents. Areal bone mineral density (BMD, g/cm(2)) and volumetric bone mineral apparent density (BMAD, g/cm(3)) were determined for lumbar spine and proximal femur using the Hologic QDR 2000 for white subjects and the QDR 4500 for the others; the instruments were cross-calibrated from data on 20 volunteers. Lumbar spine BMD was 18% higher in AA ( p0.001) and 4% lower in Somali ( p = 0.147) than white women. Femoral neck BMD was 27% higher in AA women but also 11% greater in Somali women (both p0.001) compared with whites. Lumbar spine BMD was 6% higher ( p = 0.132) and femoral neck BMD 21% higher ( p0.001) in AA than white men. No Somali men were studied. After correcting for bone size differences, both lumbar spine ( p0.01) and femoral neck BMAD ( p0.001) were greater for Somali than white women, but the difference between Somali and AA women persisted. Lumbar spine and femoral neck BMAD values also remained significantly greater for AA women (both p0.001) and men ( p0.05; p0.001) compared with whites. Weight was associated with BMAD at both skeletal sites in all groups, but adjustment for differences in weight did not reduce the discrepancy in BMAD values between Somali and AA women or between the latter group and whites. This heterogeneity among different ethnic groups of African heritage may provide an opportunity for research to better explain race-specific differences in bone metabolism.

It has generally been held that estrogen and testosterone are the major sex steroids regulating bone metabolism in women and men, respectively. However, the description of several "experiments of nature" led to a reconsideration of this notion. Thus, a male carrying homozygous mutations in the ER-alpha gene and two males with homozygous mutations in the aromatase gene had osteopenia, unfused epiphyses, and elevated indices of bone turnover. Though these findings indicated that estrogen plays a role in regulating the male skeleton, they left unresolved the issue of whether estrogen acted on the male skeleton mainly to enhance bone mass acquisition during growth and maturation, or whether it also acted to retard bone loss in aging individuals. To address this issue, several cross-sectional observational studies have related bone mineral density (BMD) to sex steroids in elderly men, and found that estrogen correlated better than testosterone with BMD. In addition, recent longitudinal studies from our group indicate that bioavailable estrogen correlated better than testosterone both with the gain in BMD in young men and with loss of BMD in elderly men. These observational studies do not, however, prove causality, which requires direct interventional studies. Thus, we eliminated endogenous testosterone and estrogen production in 59 elderly men (mean age 68 years), studied them first under conditions of physiologic testosterone and estrogen replacement, and then assessed the impact on bone turnover of withdrawing both testosterone and estrogen, withdrawing only testosterone, only estrogen, or continuing both. We found that estrogen played the major role in regulating bone resorption in these men, and that both estrogen and testosterone were important in maintaining bone formation. Collectively then, these findings indicate that estrogen plays a dominant role in regulating the male skeleton.

The epidemiology of bone loss in populations of Asian heritage is still poorly known. This study compared the skeletal status of a convenience sample of 396 Southeast Asian immigrants (172 Vietnamese, 171 Cambodians and 53 Laotians) residing in Rochester, Minnesota in 1997 with 684 white subjects previously recruited from an age-stratified random sample of community residents. Areal bone mineral density (BMD, g/cm2) and volumetric bone mineral apparent density (BMAD, g/cm3) were determined for lumbar spine and proximal femur using the Hologic QDR 2000 instrument for the white population and the QDR 4500 for Southeast Asian subjects; the machines were cross-calibrated from data on 20 volunteers. Lumbar spine BMD was 7% higher in white than Southeast Asian women ( p < 0.001), and similar results were observed for the femoral neck; lumbar spine BMD was 12% higher in white than nonwhite men ( p < 0.001). Race-specific discrepancies were reduced by calculating BMAD: for premenopausal women, lumbar spine and femoral neck differences between whites and Southeast Asians were eliminated; for postmenopausal women the lumbar spine differences persisted ( p < 0.0001), while femoral neck BMAD was actually higher for Southeast Asians. There were no race-specific differences in femoral neck BMAD among men of any age ( p= 0.312), but lumbar spine BMAD was less for younger ( p= 0.042) but not older ( p= 0.693) Southeast Asian men. There were differences among the Southeast Asian subgroups, but no clear pattern emerged. Predictors of lumbar spine BMAD in Southeast Asian women were age ( p < 0.001), weight ( p= 0.015) and gravidity ( p= 0.037). Even after adjusting for bone size using BMAD, 32% and 9% of Southeast Asian women and men, respectively, would be considered to have osteoporosis at the femoral neck and 25% and 4%, respectively, at the lumbar spine. These findings indicate a need for culturally sensitive educational interventions for Southeast Asians and for physicians to pursue diagnosis and treatment to prevent osteoporosis-related disabilities in this population.

Transforming growth factor-beta (TGF-beta) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-beta on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-kappaB ligand (RANK-L) (50 ng/ml), TGF-beta1 (0.01-5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-beta1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-beta1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-beta1 resulted in a significant increase in the percentage of RANK+ RAW cells (P < 0.05), as well as an increase in the fluorescence intensity per cell (P < 0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-beta directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression.

There is growing awareness that therapeutic decision-making may be confounded by discrepancies in the prevalence of osteoporosis by World Health Organization criteria when bone density is measured at different skeletal sites. To explore this issue, we measured bone density at a variety of skeletal sites in a population-based sample of 348 men (age 22-90 years) and 351 women (age 21-93 years). Men had greater areal bone mineral density (BMD, g/cm2) than women at almost every subregion on total body, anteroposterior (AP) and lateral lumbar spine, proximal femur and forearm scans by dual-energy X-ray absorptiometry. However, adjustment for height or, where possible, calculation of bone mineral apparent density (BMAD, g/cm3) reduced or eliminated these differences. In addition, three different patterns of change in bone density over life were observed at the various skeletal sites as judged from cross-sectional data: no apparent age-related bone loss (e.g., AP spine BMD in men); linear bone loss over life in both sexes beginning in young adulthood (e.g., femoral neck BMD); and bone loss beginning around the time of menopause or a comparable age in men (e.g., midradius BMD). The various adjustments for bone size and the different patterns of age-related change in bone density had profound effects on the estimated prevalence of osteoporosis by World Health Organization criteria, which ranged from 2% to 45% among postmenopausal women and from 0 to 36% among men 50 years of age and older depending upon the skeletal parameter that was assessed. These observations emphasize the difficulties involved in attempts to standardize BMD scores and definitions of osteoporosis for clinical use.

Intestinal calcium absorption decreases with aging, but it is unclear whether this is attributable to an age-related intestinal resistance to 1,25-dihydroxyvitamin D [1,25(OH)2D] action. Thus, we assessed the in vivo dose response of active intestinal calcium absorption to a broad range of circulating 1,25(OH)2D levels in elderly [age (mean +/- SD), 72.5+/-3.0 yr] vs. young women (age, 28.7+/-5.3 yr; n = 20 per group), who were stratified into 5 subgroups: group 1 was given a high calcium intake of 75 mmol/day, suppressing 1,25(OH)2D levels; group 2 was given a normal calcium diet of 15-30 mmol/day, representing basal 1,25(OH)2D levels; group 3 was given a low-calcium diet of 5 mmol/day to stimulate endogenous 1,25(OH)2D production; group 4 was given the low-calcium diet plus 1 microg/day 1,25(OH)2D; and group 5 was given a low-calcium diet plus 2 microg/day 1,25(OH)2D. After 7 days of diet and/or 1,25(OH)2D treatment, fasting fractional calcium absorption (FCA) was assessed by a double-tracer method using stable calcium isotopes. Serum 1,25(OH)2D and vitamin D-binding protein levels were measured concurrently, and the free 1,25(OH)2D index [molar ratio of 1,25(OH)2D to DBP] was calculated. FCA was significantly correlated with the free 1,25(OH)2D index in the young (R = 0.63, P = 0.003) but not in the elderly women (R = 0.27, P = 0.25). Moreover, the slope of the relationship between FCA and free 1,25(OH)2D index (representing intestinal sensitivity to 1,25(OH)2D) was significantly greater in the young (compared with the elderly) women [mean +/- SEM, 0.15+/-0.04 (young) vs. 0.03+/-0.02, elderly, P = 0.03]. Thus, using an experimental design that allowed us to assess FCA over a wide range of 1,25(OH)2D levels, we demonstrate that elderly women have a resistance to 1,25(OH)2D action that may contribute to their negative calcium balance, secondary hyperparathyroidism, and bone loss.

Insulin-like growth factors (IGFs) are important regulators of the activity of mature osteoblasts, but their effects on osteoprogenitor cells in human bone marrow stroma are unclear. In this study, we assessed the effects of IGFs on a conditionally immortalized human marrow stromal cell line, hMS(3-4), which has the ability to differentiate to either mature osteoblasts or adipocytes. hMS(3-4) cells expressed functional receptors for IGFs as well as specific IGF-binding proteins (IGFBP-3, -4, -5, and -6). IGF treatment of hMS(3-4) cells did not alter IGFBP expression, but resulted in distinct posttranslational modifications of secreted IGFBP-3 and IGFBP-4 proteins. IGF-I, IGF-II, and their receptor-activating analogs significantly increased by 2-fold the proliferation rate of the hMS(3-4) cells, but had a more complex effect on hMS(3-4) cell differentiation. Treatment with IGFs did not affect gene expression of Cbfa1 or peroxisome proliferator-activated receptor gamma2 (transcription factors involved in commitment to osteoblast and adipocyte pathways, respectively), alkaline phosphatase, type I collagen, and osteocalcin (markers of the osteoblast lineage), or lipoprotein lipase and adipsin (markers of the adipocyte lineage) and did not change alkaline phosphatase activity or type I collagen and osteocalcin protein relative to total protein production. In contrast, IGFs significantly increased type I collagen expression in differentiated hMS(3-4) cells as well as mature osteoblasts and promoted lipid accumulation in differentiated adipocytes. In summary, hMS(3-4) cells express essential components of the IGF system and respond to IGF treatment with increased proliferation. There was no evidence for IGFs directly modulating the commitment of hMS(3-4) cells to either osteoblast or adipocyte pathways, and their effects on differentiation within these lineages were dependent on the stage of cell maturation.

Osteoporosis is a serious complication of systemic glucocorticoid use. However, while glucocorticoids increase bone resorption in vitro and in vivo, the mechanism(s) of this effect are at present unclear. Recent studies have identified the osteoprotegerin (OPG) ligand (OPG-L) as the final effector of osteoclastogenesis, an action that is opposed by the soluble neutralizing receptor, OPG. Thus, we assessed glucocorticoid regulation of OPG and OPG-L in various human osteoblastic lineage cells using Northern analysis, RT-PCR, and ELISA. Dexamethasone inhibited constitutive OPG messenger RNA (mRNA) steady-state levels by 70–90% in primary (MS) and immortalized stromal cells (hMS), primary trabecular osteoblasts (hOB), immortalized fetal osteoblasts (hFOB), and osteosarcoma cells (MG-63). In hFOB cells, dexamethasone inhibited constitutive OPG mRNA steady-state levels in a dose- and time-dependent fashion by 90%, and also suppressed cytokine-stimulated OPG mRNA steady-state levels. Dexamethasone-induced inhibition of OPG mRNA levels was not affected by the protein synthesis inhibitor, cycloheximide, and was shown to be due to inhibition of OPG gene transcription using a nuclear run-on assay. Moreover, dexamethasone also dose dependently (10−10m–10−7m) inhibited constitutive OPG protein concentrations in the conditioned medium of hFOB cells from 2.59 ± 0.02 ng/ml (control) to 0.30 ± 0.01 ng/ml (88% inhibition; P < 0.001 by ANOVA). Concurrently, dexamethasone stimulated OPG-L mRNA steady-state levels in MS and hFOB cells by 2- and 4-fold, respectively. Treatment of murine marrow cultures with conditioned medium harvested from dexamethasone-treated MG-63 cells increased tartrate-resistant acid phosphatase (TRAP) activity by 54% (P < 0.005) compared with medium harvested from control-treated cells (in the presence of OPG-L and macrophage colony-stimulating factor). Moreover, dexamethasone (10−8m) promoted osteoclast formation in vitro, as assessed by a 2.5-fold increase of TRAP activity in cell lysates (P < 0.001) and the appearance of TRAP-positive multinucleated cells. Our data are thus consistent with the hypothesis that glucocorticoids promote osteoclastogenesis by inhibiting OPG and concurrently stimulating OPG-L production by osteoblastic lineage cells, thereby enhancing bone resorption.

This population-based study documents an increase in most types of fractures following the occurrence of a clinically recognized vertebral fracture among 820 Rochester, Minnesota, residents. During 4349 person-years of follow-up, 896 new fractures were observed. Relative to incidence rates in the community, there was a 2.8-fold increase in the risk of any fracture, which was greater in men (standardized incidence ratio (SIR), 4.2; 95% CI, 3.2-5.3) than women (SIR, 2.7; 95% CI, 2.4-3.0). The estimated cumulative incidence of any fracture after 10 years was 70%. The greatest increase in risk was for subsequent fractures of the axial skeleton, in particular a 12.6-fold increase (95% CI, 11-14) in additional vertebral fractures. There was a lesser increase in most limb fractures, including a 2.3-fold increase (95% CI, 1.8-2.9) in hip fractures and a 1.6-fold increase (95% CI, 1.01-2.4) in distal forearm fractures. There was a slightly greater association with distal forearm fractures among those whose first vertebral fracture occurred before age 70 years but a similar relationship with hip fractures, including cervical and intertrochanteric hip fractures separately, regardless of age at the initial vertebral fracture. There was also an equivalent increase in subsequent fracture risk whether the initial vertebral fracture was attributed to severe or moderate trauma. These data show that vertebral fractures represent an important risk factor for fractures in general, not just those of the spine and hip.

Recent studies have identified osteoprotegerin ligand (OPG-L) as the essential factor required for osteoclastogenesis, and that the effects are prevented by its soluble receptor, osteoprotegerin (OPG). However, there are limited data at present on the regulation of OPG-L expression in human osteoblastic cells by other cytokines. Because interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and IL-6 all increase osteoclastogenesis, we assessed whether OPG-L mRNA steady-state levels were regulated by these cytokines in human osteoblastic cells. By northern analysis, IL-1beta (5 nmol/L) and TNF-alpha (9 nmol/L) increased OPG-L mRNA steady-state levels by up to two- to three-fold in normal marrow stromal cells (MS), an immortalized marrow stromal cell line (hMS), and the osteosarcoma cell line, MG-63, whereas IL-6 (2 nmol/L, with or without its soluble receptor) had no effect on OPG-L mRNA levels in any of these cells. IL-1beta and TNF-alpha increased OPG-L mRNA steady-state levels in the normal MS cells and the hMS cell line in a time- and dose-dependent fashion by up to 4.1-fold and up to 2.6-fold, respectively. Our data are thus consistent with the hypothesis that the proinflammatory and bone-resorbing cytokines, IL-1beta and TNF-alpha, but not IL-6, may stimulate osteoclastogenesis by inducing the expression of OPG-L.

Although osteoporosis has traditionally been considered a disease of women, men also incur substantial bone loss with aging, and elderly men have age-specific hip fracture incidence rates and vertebral fracture prevalence rates that are at least half those in women. Early postmenopausal bone loss (which results in the syndrome of type I osteoporosis) is due to the direct skeletal consequences of estrogen deficiency, manifested by an increase in bone resorption without an adequate increase in bone formation. Recent evidence indicates that even late postmenopausal bone loss (type II or ‘senile’ osteoporosis) in women may be due to estrogen deficiency. In particular, the late consequences of estrogen deficiency in elderly women result in abnormalities in calcium homeostasis and increases in parathyroid hormone secretion, leading to increased bone resorption and bone loss. The etiology of bone loss in aging men has remained relatively unclear. Recent evidence from a male deficient in estrogen receptor-alpha and in two males with aromatase deficiency indicate that estrogen may play a significant role in bone metabolism in men. Moreover, several large epidemiologic studies have found that bone mineral density correlates better with serum estrogen than testosterone in aging men. Thus estrogen deficiency may lead to bone loss in men.

Bone morphogenetic proteins (BMPs) induce differentiation of osteoblast and chondroblast lineage cells from uncommitted mesenchymal precursors. Because estrogen has potent osteochondrogenic actions, we investigated its effect on BMP production in two estrogen-responsive, human immortalized cell lines (hFOB/ER3 and hFOB/ER9) that display the mature osteoblast phenotype. These cell lines were produced by stable transfection of the estrogen receptor (ER) gene into immortalized fetal osteoblasts at low ( approximately 800 ER/ nucleus) and at high ( approximately 3, 900 ER/nucleus) levels, respectively. As assessed by reverse transcriptase PCR, treatment with 17beta-estradiol (10(-)10 - 10(-)7 M) increased steady-state levels of BMP-6 mRNA dose dependently by twofold in the hFOB/ER3 cells and by over threefold in the hFOB/ER9 cells. Messenger RNA levels for transforming growth factors-beta1 and -beta2 and BMPs-1 through -5 and -7 levels were unchanged. The results were confirmed by sequence determination of the PCR product and by Northern blot analysis for total RNA. 17beta-estradiol increased BMP-6 protein production sixfold by Western analysis. Cotreatment with antiestrogens (ICI 182,780 or 4-hydroxytamoxifen) antagonized the effects of 17beta-estradiol. These data suggest that some of the skeletal effects of estrogen on bone and cartilage may be mediated by increased production of BMP-6 by osteoblasts.

Insulin-like growth factors (IGF)-I and IGF-II are produced by osteoblasts and are important paracrine/autocrine regulators of osteoblast proliferation and differentiation. Estrogen has been reported to increase gene expression of IGF-I in rodent osteoblasts. However, because species differences have been demonstrated in expression of various aspects of the IGF system in bone cells, it is not known whether this action also occurs in human osteoblasts. Thus, we assessed the effects of estrogen treatment on IGF-I and IGF-II gene expression in vitro in a recently developed human fetal osteoblast cell line that has high levels of estrogen receptors. As assessed by a quantitative reverse transcriptase-polymerase chain reaction method, treatment of hFOB/ER9 cells with 17beta-estradiol (E2) increased steady state levels of IGF-I mRNA in a time- and dose- dependent fashion with a maximal increase of 319% +/- 33% (P0.01) of control occurring after treatment with 10(-7) M E2 for 48 hours. In contrast, E2 did not alter steady state levels of IGF-II mRNA. The pure (type 2) antiestrogens ICI 182,780 (10(-7) M) and ICI 164,384 (10(-6) M) blocked the E2- induced increase in IGF-I mRNA levels. Interestingly, 4-hydroxytamoxifen (10(-7) M), a documented pure antiestrogen in reproductive tissues, also increased IGF-I mRNA to levels similar to those observed in E2-treated cells. Since E2 was shown to mediate its effects on some target genes through a cAMP-dependent pathway, we studied the interaction between E2 and agents that are known to increase intracellular cAMP. Forskolin (10(-8) M) and dibutyryl cAMP (10(-3) M) increased IGF-I mRNA levels sixfold, and cotreatment with E2 did not affect these changes, consistent with a possible mediation of the estrogen effect on IGF-I gene expression by cAMP. We conclude that in human osteoblastic cells, the IGF-I gene is a target for estrogen action, suggesting that IGF-I may mediate part of the effects of estrogen in human bone.

Estrogen (E2) has been shown to prevent bone loss among postmenopausal women. The molecular mechanism(s) by which this is accomplished is not clear. The discovery of E2 receptor (ER) in osteoblasts and osteoclasts has implicated these cells as direct targets for E2. Previous studies on the effects of E2 on osteoblastic cells in vitro or in organ culture present conflicting results, possibly due to heterogeneity in cell types, stage of differentiation, ER levels, and/or species differences. The effects of E2 on gene expression during various stages of human osteoblast cell differentiation has not been investigated extensively. In this study we employed a newly developed human fetal osteoblastic cell line (hFOB/ER9) that contains high levels of ER to examine the effects of E2 on osteoblast proliferation and differentiation. The basal levels and E2 effects on the expression of various extracellular matrix proteins were also characterized throughout different stages of differentiation. These stages include a proliferative/relatively undifferentiated stage (day 6), a matrix maturation stage (days 10-14), and a mineralization/calcified nodule stage (day 18). During the stage of rapid cell proliferation, E2 treatment of hFOB/ER9 cells resulted in a dose-dependent decrease in [3H]thymidine incorporation to a maximum of 72% compared to the vehicle control value. Treatment of hFOB/ER9 cells with 10(-9) M E2 for 48 h resulted in an increase in alkaline phosphatase (AP) activity throughout cell differentiation. The magnitude of AP induction varied from approximately 200-500%. In contrast, E2 decreased osteocalcin protein levels to a minimum of 54% compared to the vehicle control value. The steady state messenger RNA levels for AP increased and osteocalcin decreased after E2 treatment, similar to the responses observed at the protein level. At all stages, there was little or no effect of E2 on type I collagen protein levels or osteonectin steady state messenger RNA levels. The E2 responses on hFOB/ER9 cell matrix protein expression and cell proliferation were mediated through the ER, as cultures cotreated with a 100-fold molar excess of a type II anti-E2 (ICI 182,780) abrogated these effects. These results support the hypothesis that E2 does have an effect on osteoblastic differentiation by decreasing hFOB/ER9 cell proliferation and differentially regulating extracellular matrix expression.

Insulin-like growth factors (IGFs) and their specific regulatory binding proteins (IGFBPs) are postulated to play a key role in bone metabolism. To date, IGFBP-2 through -6 have been characterized in bone cell systems. In this study we focused on IGFBP-1. Primary cultures of normal human osteoblasts derived from trabecular bone (hOB cells) expressed low levels of IGFBP-1 messenger RNA (mRNA), as determined by Northern analyses. Treatment of hOB cells with 1 microM cortisol or 100 nM dexamethasone for 20 h stimulated IGFBP-1 mRNA expression 5-fold and increased levels of immunoassayable IGFBP-1 in the conditioned medium 3-fold. Estradiol and progesterone had no effect. IGFBP-1 expression was not observed in U-2, TE-85, or MG-63 human osteosarcoma cell lines or in normal human fibroblasts. Insulin (1-100 nM) potently inhibited both basal and glucocorticoid-stimulated IGFBP-1 expression in hOB cells. Insulin had little or no effect on steady state levels of the other IGFBP mRNA. A monoclonal antibody to the insulin receptor blocked insulin binding to insulin receptors and completely prevented insulin-induced suppression of IGFBP-1. In summary, we have documented IGFBP-1 mRNA and protein expression in normal nontransformed human osteoblastic cells. This expression was stimulated by glucocorticoids and inhibited by insulin in a manner similar to IGFBP-1 regulation in hepatocytes. Insulin acts through insulin receptors on hOB cells. We postulate that IGFBP-1 produced by osteoblasts in vivo can modulate local actions of IGF on bone formation in response to changes in glucocorticoid and insulin concentrations.

Serum parathyroid hormone (PTH) and bone resorption increase in elderly women and contribute to age-related bone loss. Whether these abnormalities are caused by calcium deficiency resulting from age-related decreases in absorption and renal conservation is unclear. We studied 28 normal elderly women (mean +/- SD, age 69.3 +/- 2.7 yr) who were maintained for 3 yr on usual calcium intake levels (20.4 +/- 7.2 mmol/day [815 +/- 289 mg/day]; n = 15) (known as the usual calcium group) or high calcium intake levels (60.4 +/- 6.5 mmol/day [2414+/260 mg/day]; n = 13) (known as the high calcium group) and a reference group of 12 normal young adult women (age 30.1 +/- 4.4 yr), whose calcium intake was 23.0 +/- 4.8 mmol/day (918 +/- 193 mg/day) (known as the young group). Serum PTH was measured every 2 h, and urinary excretion of deoxypyridinoline (Dpd), a new marker for bone resorption, was measured in 4 h collections. Parathyroid gland secretory capacity was assessed during induced hypocalcemia. The mean 24 h serum PTH was 40% lower (P0.001), and the mean 24 h urinary Dpd was 35% lower (P0.005) in the high than in the usual calcium group. Mean parathyroid gland secretory capacity also was 47% lower (P0.005) in the high calcium group than in the usual calcium group. However, the usual calcium group had a mean 24 h serum PTH level that was 70% higher (P0.001) and a mean 24 h urinary Dpd level that was 30% higher (P0.005) than the young group, whereas the high calcium group was indistinguishable from the young group. Thus, failure of elderly women to increase their calcium intake to offset age-related increases in calcium requirement contributes substantially to their development of increased parathyroid activity and increased bone resorption, whereas a high calcium intake can reverse both abnormalities.

Recent studies in rodents and in human in vitro systems suggest that the action of estrogen on bone is modulated by various bone-resorbing cytokines produced by osteoblasts, bone marrow cells, or both, but results among studies have been conflicting. Thus, we studied 20 untreated women (controls) and 20 women who had received estrogen replacement therapy since the time of menopause; both groups were 5 +/- 1 yr (mean +/- SE) postmenopausal. From bone marrow aspirates, we obtained marrow plasma to evaluate the production of cytokines by marrow and trabecular bone cells in vivo and marrow mononuclear cells to assess in vitro the constitutive and lipopolysaccharide-stimulated production of cytokines and also to test the in vitro effects of 17 beta-estradiol (10(-8) mol/L). Interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-1 receptor antagonist, and IL-6-soluble receptor were measured by specific enzyme-linked immunosorbent assay. No statistically significant difference between the two groups in the level of any of these cytokines was detected in the bone marrow plasma or the conditioned medium in vitro. Furthermore, in vitro treatment with 17 beta-estradiol did not affect basal or lipopolysaccharide-stimulated cytokine production. Thus, our data suggest that for normal post-menopausal women, mediation of the effects of estrogen by a single cytokine is unlikely. Either multiple cytokines are involved and small changes in these are difficult to detect in clinical investigative studies, or other mechanisms are operative.

Estrogen deficiency induced by menopause leads to an increase in bone resorption that is not compensated for by a comparable increase in bone formation, resulting in excessive bone loss. Clinically, estrogen replacement reverses these processes, but the mechanisms by which this takes place are not completely understood. Both osteoclasts and osteoblasts contain functional estrogen receptors and, therefore, may be directly involved in these responses. Because both osteoclasts and osteoblasts secrete transforming growth factor-beta (TGF beta), and because 17 beta-estradiol (E2) treatment increases TGF beta production by osteoblast-like cells in vitro, we have investigated the possibility that E2 also may increase the production of TGF beta by isolated osteoclasts in vitro. Highly purified avian osteoclasts were treated with either vehicle or E2, and TGF beta protein accumulation in culture was measured by bioassay. Although an E2 dose-dependent increase in TGF beta protein accumulation in osteoclast-conditioned medium was measured at 4 h of treatment, a steroid dose-dependent decrease in the accumulation of active TGF beta was measured after 18 h of estrogen treatment. The steroid specificity of the increased TGF beta accumulation was confirmed by demonstrating that the E2-induced increase in TGF beta protein levels in the medium was inhibited by cotreatment with a specific E2 antagonist. Interestingly, E2 treatment induced a TGF beta isoform change from TGF beta 2 to predominantly TGF beta 3. Thus, the data suggest that a direct action of E2 on osteoclasts to lower resorption activity may be mediated by autocrine/paracrine production and activation of TGF beta, perhaps including modulation of specific isoform production.

PTH has been postulated to play a role in both nocturnal and age-related increases in bone resorption. We tested this hypothesis directly in 10 young (ages 24-35 yr) and 10 elderly (ages 71-78 yr) normal women by measuring the cross-linked N-telopeptide of type I collagen (NTx), a marker for bone collagen breakdown, in 4-h urine collections before and during suppression of PTH secretion by a 24-h iv infusion of calcium. Serum ionized calcium and PTH levels were also measured every 2 h before and during the infusion. In both groups of women, serum PTH levels and urinary NTx excretion followed a circadian pattern before calcium infusion (analysis of variance, P = 0.0001) with peaks in the afternoon and at night for PTH and at night for urinary NTx. During the calcium infusion, the nocturnal urinary NTx excretion peak persisted (P = 0.0001), despite elimination of both PTH peaks. Urinary 24-h NTx excretion (nanomoles per millimoles of creatinine) at baseline was higher in the elderly women (mean +/- SEM, 25.7 +/- 2.1) than in the young women (19.3 +/- 1.7) (P < 0.01), and the decrease during calcium infusion was greater (7.5 +/- 1.9 vs. 4.1 +/- 1.5, P < 0.05). Therefore, the increase in serum PTH levels with age is one of the major factors responsible for the age-related increase in bone resorption. PTH does not mediate the circadian pattern of bone resorption but does play a role in setting the absolute level of bone resorption at which this pattern occurs.

Because the osteoporosis occurring in chronic cholestatic liver disease (CCLD) is associated with decreased bone formation and is reversible by liver transplantation, substances retained in plasma during cholestasis may impair osteoblast function. This hypothesis was tested using a new bioassay that measures plasma mitogenic activity (PMA) for normal human osteoblast-like (hOB) cells. In 29 jaundiced patients, mean PMA was 56.4% (P < 0.001) of that in 29 age- and sex-matched normal subjects, and the decrease in PMA was similar in the 14 with CCLD and the 15 with other causes of jaundice. Bile acids and bilirubin are the two major groups of products retained during cholestasis. The common conjugated bile acids and bilirubin were added to normal human plasma in concentrations simulating those found in patients with CCLD. Various bile salts had no effect on PMA whereas unconjugated bilirubin decreased PMA in a dose-dependent fashion (r = -0.98, P < 0.0001) without affecting cell viability. Relatively selective removal of bilirubin from the plasma by photobleaching normalized the decreased PMA in five jaundiced patients but produced no apparent change in five normal subjects. These data support the hypothesis that hyperbilirubinemia or possibly other photolabile substances impair osteoblast proliferative capacity and thus may play a major role in the pathogenesis of the osteoporosis associated with CCLD.

In a 4-year controlled, prospective trial, histomorphometric analysis was used to compare the tissue-level skeletal effects of fluoride therapy in 43 postmenopausal women (75 mg NaF/day) with those of 35 matching placebo subjects; all subjects received 1500 mg/day elemental calcium supplement. In addition to an initial, baseline biopsy, a second biopsy was obtained after 6, 18, 30 or 48 months. Measurements were made on a third biopsy obtained from 8 subjects following at least 72 months of fluoride therapy. The change in cancellous bone volume or trabecular thickness in fluoride-treated subjects was not different from a change in placebo-treated subjects. However, paired analysis in the fluoride-treated subjects indicated that bone volume was increased between the first and second biopsies (p < 0.005). Both osteoid length and width were significantly increased in fluoride compared with placebo subjects; however, only the osteoid surface increased linearly (r = 0.63, p < 0.001). The mineral apposition rate and relative tetracycline-covered bone surface were not different between fluoride and placebo treatment, although they were decreased in both groups in the second biopsy. The tetracycline-covered bone surface returned to normal in the third biopsy. Definitive evidence for osteomalacia is a prolonged mineralization lag time, which following fluoride treatment was found to be increased 9-fold in the second biopsy and 4-fold in the third biopsy. Further evidence for osteomalacia was increased osteoid thickness by 6 months, evidence of focal areas of interstitial mineralization defects, and broad tetracycline labels of low fluorescence intensity. In the third biopsies, osteoclastic resorption was observed beneath osteoid seams. Fluoride therapy increased the cortical width compared with placebo treatment (p < 0.02), and increased the osteoid surface in Haversian canals, but did not change the osteoid width, resorption surface or cortical porosity. After an initial rise, serum fluoride levels remained constant, and the urine values fell slightly. The bone fluoride concentration rose throughout the treatment period, and was correlated with the change in osteoid-covered bone surface (r = 0.56, p < 0.001). Although we found definitive evidence for osteomalacia, the cause of the osteomalacia was not determined in this study. On the other hand, the presence of bone resorption beneath unmineralized osteoid and of osteocyte halos is suggestive of hyperparathyroidism. Thus, it is possible that the strong stimulus for bone formation brought about by fluoride therapy resulted in relative calcium deficiency.

Osteoporosis is one of the major problems facing the aging population. The two most important variables in evaluating patients with osteoporosis, either from the standpoint of research studies or in their clinical management, are the prevailing level of bone mineral density (BMD) and the level of bone turnover.

Scientists are placing increasing emphasis on developing immortalized cells in culture in order to achieve rapidly proliferating, cell systems with extended lifetimes (e.g., increased population doublings) and normal phenotypic characteristics. As shown in Figure 1, cells undergo a differentiation process, function in the differentiated state for a period of time, then undergo an aging process (senescence) and cell death. Cell senescence can be defined as the failure of cells to maintain appropriate functions for life including cell replication, often resulting in programmed cell death (apoptosis) [1, 2]. Cell fusion studies suggest that there are processes in senescent cell nuclei that block DNA replication, maintaining cells in the late G1 stage of the cell cycle, whereas normal cell nuclei stop at the early G1 stage. Current methods to immortalize cells can delay but not prevent senescence (see Fig. 1) [1, 2]. Immortalization appears to be an intermediate step in cell transformation, the latter resulting in loss of many cell properties and a more permanent immortalization.

Recent reports of increases in serum 1,25-dihydroxyvitamin D [1,25(OH2)D] concentration with aging despite no changes or decreases in calcium absorption suggest that elderly women have intestinal resistance to vitamin D action. Thus, in 15 young adult (30 +/- 1 year) and 15 elderly (74 +/- 1 year) women (mean +/- SE), we assessed the responsiveness of intestinal calcium absorption to increases in circulating 1,25(OH)2D induced by 4 days of an experimental diet (150 mg calcium and 1600 mg phosphorus daily). True fractional calcium absorption (FCA) (44Ca mixed with food and 42Ca given intravenously, then their ratio in urine measured by mass spectrometry) was determined. Baseline serum intact parathyroid hormone (PTH) concentration was higher in the older women (P = 0.01) whereas serum 1,25(OH)2D concentration and true FCA were similar. In both groups, serum 1,25(OH)2D concentrations increased (P0.002) on the experimental diet. After 4 days on the diet, serum 1,25(OH)2D increased over baseline by 30.5 and 35.6% and, despite these increases, true FCA was 40 +/- 3 versus 40 +/- 4%/24 hours (NS between groups) in the young and elderly women, respectively. These data suggest that either elderly women have normal intestinal responsiveness to vitamin D or that the resistance to it is too mild to be detected by these methods.

The decrease in estrogen levels that follows the onset of menopause results in rapid bone loss and osteoporosis. The major effect of estrogen deficiency on bone metabolism is an increase in the rate of bone resorption, but the precise mechanism by which this occurs remains unresolved. A recently developed technique for the isolation of avian osteoclasts has been modified to obtain highly purified multinucleated cells from human giant cell tumors. These osteoclast-like cells have been examined for evidence of estrogen receptors (ERs) and responses to 17 beta-estradiol (17 beta-E2). Analysis of giant-cell RNA demonstrated expression of ER mRNA. Furthermore, immunoblot analysis revealed that the giant cells contained a 66-kDa protein that was recognized by a monoclonal antibody specific for the human ER. When isolated multinucleated cells were cultured on slices of bone, there was a dose-dependent decrease in resorption in response to treatment detectable at 10 pM 17 beta-E2. Treatment with 10 nM 17 alpha-estradiol or vehicle (control) did not inhibit resorption. Moreover, the multinucleated cells isolated from these tumors had decreased mRNA levels for cathepsin B, cathepsin D, and tartrate-resistant acid phosphatase (TRAP) as well as secreted cathepsin B and TRAP enzyme activity in response to treatment with 10 nM 17 beta-E2. In contrast to these data, no change in gene expression was detected in mononuclear cells from these tumors in response to 17 beta-E2 treatment. These data support the proposition that human osteoclasts are target cells for estrogen and that estrogen can inhibit bone resorption by human osteoclasts.

Transforming growth factor-beta (TGF beta) is a multifunctional growth factor in bone that is secreted as a latent complex and must be activated in order to influence cellular activity. We have investigated the influence of dexamethasone (Dex; a potent glucocorticoid) on the secretion and activation of latent TGF beta by normal human osteoblast-like cells (hOBs). Dex had no significant effect on TGF beta mRNA or total protein production, but treatment with Dex resulted in a steroid dose-dependent activation of up to 90% of the TGF beta produced by the hOBs. Dex-treated hOBs activated multiple latent forms of TGF beta. Conditioned medium from Dex-treated hOBs retained the ability to activate latent TGF beta when incubated at 37 C in the absence of cells. Unlike other cell systems, Dex-induced hOB-mediated TGF beta activation did not involve binding to the mannose-6-phosphate receptor. However, the activation was prevented by treatment of hOB cells with microtubual disrupting agents, by the addition of protease inhibitors, or by weak base treatment of the medium. Dex treatment of hOBs was shown to induce a dose-dependent increase in the mRNA levels of cathepsin-B and -D and in the levels of cathepsin-B protein secreted by the cells. Taken together, these data suggest that Dex treatment of hOBs induces the production and secretion of lysosomal proteases that, when secreted, activate latent TGF beta which is secreted by the hOB cells. There is evidence for an involvement of more than one type of protease in this activation process. This activation of TGF beta may, therefore, play a role in glucocorticoid regulation of bone cell functions. Furthermore, TGF beta is most likely involved in autocrine and paracrine effects on bone cells.

The aims of the study were to evaluate the use of bone-specific biochemical markers of turnover in type I osteoporosis, to test for evidence of heterogeneity of bone turnover in this condition, and to attempt to devise an ‘uncoupling index’ by using the relationship between bone-specific biochemical markers of bone formation and bone resorption. In women with type I osteoporosis (mean age 64 years, SD 5;n=63) the mean level of serum osteocalcin, a specific biochemical marker of bone formation, was 9.9 ng/ml (SD 2.0), which was higher than the level in normal postmenopausal women (mean age 65 years, SD 6;n=8.9 ng/ml (SD 2.0;p

We developed a competitive chemiluminoimmunoassay of osteocalcin that is similar to radioimmunoassay but uses acridinium-ester-labeled antigen instead of 125I-labeled osteocalcin; a second antibody immobilized on plastic beads is used to separate free and bound fractions. There was good correlation of the new chemiluminoimmunoassay (y) with a polyclonal antiserum (R102) radioimmunoassay (x) used in many previous clinical studies (r = 0.96, y = 0.968x + 2.69, Sy/x = 0.029, n = 86). The new assay recognized both the intact and the small fragment of osteocalcin in plasma and detected decreases of them (total, approximately 47%) after a 24-h infusion of parathyroid hormone. Patients with primary hyperparathyroidism had increased concentrations of intact osteocalcin. Children had higher concentrations of osteocalcin than adults did. Healthy women had greater osteocalcin concentrations at ages 50-70 years than earlier. Inverse correlations of bone mineral density and osteocalcin were found in healthy women and in women with osteoporosis.

There is a need to identify vertebral fractures from radiographs taken at a single point in time, but considerable controversy surrounds the methods to be used. We extended a data set to comprise baseline radiographs of the thoracic and lumbar spine on an age-stratified random sample of 762 Rochester, Minnesota, women and used revised methods to define vertebral deformities morphometrically. Changes in the method of measuring vertebral heights, changes in the source of normal values for vertebral measurements and changes in the criteria for assessing vertebral deformity had little impact on estimated prevalence and incidence in this population. The prevalence of any vertebral deformity was estimated at 25.3 per 100 Rochester women aged 50 years and over (95% CI, 22.3-28.2), while the incidence of a new deformity in this group was estimated at 17.8 per 1000 person-years (95% CI, 16.0-19.7). Projected nationally, these data suggest that over 500,000 white women in the United States develop vertebral deformities for the first time each year and that over 7 million white women aged 50 years and over might be affected at any given time. These estimates are limited by the absence of a reliable 'gold standard' with which to determine false positive and false negative rates associated with this or any other morphometric method. Information on the health consequences of vertebral deformities of various sorts would be most helpful in choosing between alternative approaches to defining them.

The influence of reproductive factors on bone mass at six skeletal sites was assessed in an age-stratified random sample of white women residing in Rochester, Minnesota. After age-adjustment, whether or not women had ever breastfed, total duration of breastfeeding and duration of breastfeeding per child were not associated with reduced bone mineral, but breastfeeding for more than 8 months was associated with greater bone mineral at some sites. There were no consistent effects on bone mineral, after adjusting for age, of gravidity or parity, age at menarche, age at first delivery, use of oral contraceptives or estrogen replacement therapy, various sex hormones, nor any of the other reproductive factors assessed. There was a strong protective effect of obesity, which was also correlated with a number of the reproductive variables. While animal studies suggest that pregnancy and lactation may be associated with calcium loss from the skeleton, these data indicate that such factors have little long-term impact on bone mass in humans and little potential for identifying women at high risk of osteoporosis later in life.

Although it has been recognized for many years that oestrogen is a key component in the maintenance of normal bone balance, the mechanisms by which oestrogen exerts its influence have remained unresolved. Recent identification of oestrogen receptors in both bone-forming osteoblasts and bone-resorbing osteoclasts has opened up exciting new areas of research on the direct effects of oestrogen on both osteoblasts and osteoclasts. This review presents an updated model for the molecular mechanisms involved in oestrogen action, the mechanism of anti-oestrogen action, and outlines recent advances in knowledge of oestrogen effects on osteoblasts, osteoclasts, and the coupling of bone resorption and bone formation.

In humans, glucocorticoids are known to have marked effects on bone metabolism and function, including the significant regulation of osteoblast cells. To aid in the understanding of the mechanism of glucocorticoid action on normal human osteoblasts (hOB), confluent cells were analyzed for the presence of glucocorticoid receptors (GR) as well as for the effects of the glucocorticoid dexamethasone (Dex) on the expression of both the rapid responding nuclear proto-oncogenes and the late responding structural genes for bone matrix proteins. The interactions between Dex and 1,25 dihydroxy vitamin D3 (1,25 D3) on the gene expression in these cells were also examined. Using a functional receptor assay, a mean of 11,600 functional nuclear bound glucocorticoid receptors (range 6,000-22,000) was measured in fifteen separate cell strains. Northern blot analysis with a cDNA probe to the human GR was used to demonstrate the presence of a 7Kb transcript which is a candidate mRNA for GR in these cells. In agreement with previous studies, treatment of the hOB cells with Dex increased the steady state mRNA levels for alkaline phosphatase (AP) but displayed little or no effect on the mRNA levels for osteocalcin (OC) and glyceraldehyde phosphate dehydrogenase (GAPDH). Interestingly, the 1,25 D3 inductions of mRNA levels for OC were blocked by Dex but enhanced for AP. The above effects of Dex on AP and OC gene expression, including the interaction with 1,25 D3, were also shown to occur at the level of protein. The effect of Dex on the mRNA levels of the nuclear proto-oncogenes c-myc, c-fos, and c-jun was also investigated, since the oncoproteins (Fos/Jun) appear to play a role in the delayed glucocorticoid regulation of structural genes. Interestingly, Dex increased the steady state levels of c-myc, c-fos, and c-jun mRNAs in nonproliferating (confluent) hOB cells by 3.5-, 10-, and 2.0-fold, respectively, over control (untreated cells) values within one h of steroid treatment. The Dex-induced mRNA levels were transient and returned to basal values within 24 h of the steroid treatment. A reduced but qualitatively similar pattern of response was found in proliferating hOB cells. The pattern of response of these genes to glucocorticoids in hOB cells mimics the response in avian liver cells but not in reproductive cells. These results support the theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucocorticoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cells.

Local secretion of insulin-like growth factor binding proteins (IGFBPs) may modulate the effects of IGF-I and IGF-II on bone cell metabolism and proliferation. Several osteoblast-derived cell lines are currently used as interchangeable models to study IGFBP production, although it is unknown whether findings in one cell line can be extrapolated to another cell line or to normal human osteoblasts. In this study, we examined by Western ligand blotting both basal and regulated secretion of IGFBPs in vitro in 1) normal human osteoblast-like (hOB) cells cultured from explants of human trabecular bone; 2) an SV40-transformed hOB (HOBIT) cell line; and 3) several human (U-2, MG-63, TE-85) and rat (ROS 17/2.8 and UMR-106-01) osteosarcoma cell lines. Constitutively, hOB and HOBIT cells produced a similar pattern of IGFBPs, while all other cell lines produced their own unique pattern of IGFBPs. The two rat cell lines differed from the human cell lines as well as from each other. The response to hormonal stimulation also varied among the cell lines. Treatment of hOB and HOBIT cells with IGF-I resulted in a 2-fold increase in medium levels of IGFBP-3; IGF-I decreased levels of 24-kilodalton (kDa) IGFBP in hOB cell-conditioned medium. In addition, IGF-I markedly increased levels of the 29/32/34 kDa IGFBP triplet in U-2 cells, but had little or no effect in the other human and rat osteosarcoma cell lines. PTH increased a 29-kDa IGFBP apparent only in UMR 106-01 cell-conditioned medium, whereas GH had no direct effect on IGFBP secretion in any of the osteoblast-like cells tested. We conclude that basal and regulated secretion of IGFBPs from osteoblast-like cells is cell-line specific. Spontaneously transformed human or rat osteoblast-like cells provide unique model systems to study features of distinct IGFBPs and their regulation; however, hOB cells and their derivatives may be more appropriate models for understanding the regulation of IGFs in human bone.

1. If gastric acid is necessary for the absorption of dietary calcium, the total absence of gastric acid secretion that occurs in pernicious anaemia could result in bone loss. To investigate this, we measured calcium absorption and bone density in 21 postmenopausal women (ages 51–76 years) with pernicious anaemia and in 24 normal postmenopausal women (ages 51–79 years). 2. Relative to the normal women, in the women with pernicious anaemia the bone mineral density of the lumbar spine was decreased by 16% (P < 0.001). 3. After adjustment for age and body weight, lumbar spine bone mineral density correlated with the serum concentration of group 1 pepsinogens (a group of proteins produced by the gastric fundus) (r= 0.61, P < 0.01). 4. Despite achlorhydria, the women with pernicious anaemia had normal true fractional calcium absorption and normal serum levels of parathyroid hormone and 1,25-dihydroxyvitamin D. 5. We conclude that gastric acid is not required for the absorption of dietary calcium. Thus, the loss of cancellous bone must be caused by some mechanism yet to be identified.

We investigated, in five cell strains per experiment, whether several cytokines known or believed to have effects on bone resorption were produced by nearly homogeneous strains of cultured normal human osteoblast-like (hOB) cells that display virtually the complete phenotype of the mature osteoblast. In unstimulated hOB cells, we detected constitutive production of interleukin-6 (IL-6) (mean +/- SE, 122 +/- 32 pg/ml) and IL-8 (135 +/- 39 pg/ml), but not of IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), or tumor necrosis factor-alpha (TNF alpha). IL-1 beta in doses from 1-100 U/ml stimulated dose-dependent increases in IL-6 (r = 0.87; P less than 0.001) and IL-8 (r = 0.95; P less than 0.001). Similar increases occurred after stimulation with TNF alpha in doses from 3-300 U/ml. IL-1 beta and TNF alpha also stimulated GM-CSF production, but only at higher doses. 17 beta-Estradiol (10(-8) M) had no significant effect on the secretion of any of these cytokines, either constitutively or after stimulation with IL-1 beta or TNF alpha. Stimulated production of IL-4 was not detected after treatment with IL-1 beta or TNF alpha, and that of TNF alpha was not detected after treatment with IL-1 beta. We conclude that IL-6, IL-8, and GM-CSF, but not IL-4 and TNF alpha, are produced by highly differentiated normal human cells of the osteoblast lineage, but their secretion is not regulated by estrogen. However, we cannot exclude the possibility that estrogen regulation of these cytokines may occur during early stages of osteoblast differentiation.

To define the role of parathyroid gland function in the pathophysiology of bone loss in type I (postmenopausal) osteoporosis, we measured serum intact parathyroid hormone (PTH) concentration by immunoradiometric assay (IRMA) and by multisite immunochemiluminometric assay (ICMA) in 63 postmenopausal osteoporotic women (PMOp) with vertebral compression fractures and in 75 age-comparable postmenopausal normal women (PMNl). Also, tetracycline-based histomorphometric indices in cancellous bone were assessed in iliac biopsy samples from 61 PMOp and 28 PMNl women. Serum PTH concentrations by IRMA were similar in PMOp and PMNl (medians, 3.92 and 3.77 pmol/l; NS) but were significantly lower in PMOp by the more sensitive ICMA (medians, 2.82 and 3.14 pmol/l; P less than 0.01). By multiple linear regression analysis, serum PTH was directly related (P less than 0.001) to activation frequency, bone resorption rate, bone formation rate, and the calculated rate of bone loss. For each unit (pmol/l) increase in serum PTH by ICMA, activation frequency increased by 1.3%/year more (P = 0.01), bone resorption rate increased by 3.9%/year more (P = 0.003), and the rate of cancellous bone loss was 2.8% greater (P = 0.0003) in the PMOp women compared with the PMNl women. Concentrations of serum estradiol, but not serum estrone, had a weak opposing effect to PTH, especially for bone formation rate. These data suggest that in PMOp the bone has increased sensitivity to the biologic effects of PTH. This may represent one of the fundamental pathophysiologic defects in PMOp and, in the setting of estrogen deficiency, may explain, in part, their greater rate of bone loss.

We have compared two assays for osteocalcin (BGP, bone gla protein), one based on a monoclonal antibody, the other based on a polyclonal serum, in an effort to better understand the factors that contribute to the wide variation reported for osteocalcin normal ranges. The two assays compared well using serum samples, with a correlation coefficient of 0.9071 (n = 48). However, the monoclonal antibody assay returns values increased over the polyclonal assay by approximately 1.5-fold. Gel permeation studies indicate that these larger values are due primarily to increased reactivity in the monoclonal assay with high- and low-molecular-weight forms of osteocalcin in plasma; both assays give similar reactions to intact osteocalcin. Analysis of samples from individuals with increased bone resorption due to parathyroid hormone administration reveals that the decrease seen in osteocalcin values after hormone infusion occurs primarily in the fraction that corresponds to intact osteocalcin. During the course of these studies, we re-evaluated the extinction coefficient for osteocalcin, arriving at the value E = 1.33 mg/ml-1, cm-1. We also observed a significant negative interference in both assays (and a commercial assay) caused by hemolysis. This interference is due to proteolysis of osteocalcin by enzymatic activity released from the lysed red cells.

Osteoporosis is one of the most important age-related diseases. Each year in the United States it causes at least 1.2 million fractures and costs 7 to 10 billion dollars. The main cause of the fractures is increased bone fragility due to low bone density, although in the elderly an increase in the frequency of falls and in the trauma produced by the falls also contributes to fractures. Low bone density in osteoporosis has multiple causes which can be grouped into the categories of low initial bone mass and bone loss due to ageing, menopause, and sporadic factors. Given the magnitude of the problem, prevention is the only cost-effective approach. Enough is known about causes of bone loss leading to osteoporosis that an effective programme of prevention can be designed. Its implementation in the population should substantially reduce the incidence of this major public health problem.