Riggs, B. Lawrence

Abstract Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. Bone morphogenetic protein 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID−1, ID−2, ID−3, and ID−4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1, STAT1, and JunB. The changes in these nuclear binding factor mRNA levels were confirmed by real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). A further three transcription factors, core binding factor β (CBFβ), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment. In summary, we have used a gene chip microarray to identify a number of BMP-2 responsive genes in hMS cells. Thus, these studies provide potential candidate genes that may induce osteoblastic differentiation or, in the case of the ID proteins, block differentiation along alternate pathways.

Abstract Several lines of evidence implicate estrogen deficiency as a cause of bone loss in elderly men. Thus, in 50 elderly men (mean age ± SD, 69.1 ± 6.0 years), we performed a randomized blinded study to assess the effect of 6 months of treatment with 60 mg/day of raloxifene (a selective estrogen receptor modulator [SERM] that has an agonist effect on bone but is not feminizing) versus placebo on bone turnover markers. The mean changes in bone turnover markers, serum sex steroid, or lipid levels with treatment did not differ between groups. However, changes in urinary cross-linked N-telopeptide of type I collagen (NTX) excretion were related directly to the baseline serum estradiol level in the raloxifene (r = 0.57; p = 0.004) but not in the placebo-treated (r = 0.15; p = 0.485) men (p = 0.015 for the difference between groups). Moreover, the men in whom NTX excretion decreased after raloxifene treatment had significantly lower baseline estradiol levels (mean ± SEM, 22 ± 2 pg/ml) than the men in whom urinary NTX excretion didn't change or increased after raloxifene therapy (30± 3 pg/ml; p = 0.03), and no such difference was found in the placebo group. Thus, raloxifene has no significant effect on bone turnover markers or lipid levels in elderly men. However, the association noted between baseline estradiol levels and the change in urine NTX excretion in the raloxifene-treated men suggests that a subset of men with low estradiol levels may respond to raloxifene or other SERMs, and further studies are needed to directly test this possibility.

Abstract In a population-based, cross-sectional study, we assessed age- and sex-specific changes in bone structure by QCT. Over life, the cross-sectional area of the vertebrae and proximal femur increased by ∼15% in both sexes, whereas vBMD at these sites decreased by 39–55% and 34–46%, respectively, with greater decreases in women than in men. Introduction: The changes in bone structure and density with aging that lead to fragility fractures are still unclear. Materials and Methods: In an age- and sex-stratified population sample of 373 women and 323 men (age, 20–97 years), we assessed bone geometry and volumetric BMD (vBMD) by QCT at the lumbar spine, femoral neck, distal radius, and distal tibia. Results: In young adulthood, men had 35–42% larger bone areas than women (p < 0.001), consistent with their larger body size. Bone area increased equally over life in both sexes by ∼15% (p < 0.001) at central sites and by ∼16% and slightly more in men at peripheral sites. Decreases in trabecular vBMD began before midlife and continued throughout life (p < 0.001), whereas cortical vBMD decreases began in midlife. Average decreases in trabecular vBMD were greater in women (−55%) than in men (−46%, p < 0.001) at central sites, but were similar (−24% and −26%, respectively) at peripheral sites. With aging, cortical area decreased slightly, and the cortex was displaced outwardly by periosteal and endocortical bone remodeling. Cortical vBMD decreased over life more in women (∼25%) than in men (∼18%, p < 0.001), consistent with menopausal-induced increases in bone turnover and bone porosity. Conclusions: Age-related changes in bone are complex. Some are beneficial to bone strength, such as periosteal apposition with outward cortical displacement. Others are deleterious, such as increased subendocortical resorption, increased cortical porosity, and, especially, large decreases in trabecular vBMD that may be the most important cause of increased skeletal fragility in the elderly. Our findings further suggest that the greater age-related decreases in trabecular and cortical vBMD and perhaps also their smaller bone size may explain, in large part, why fragility fractures are more common in elderly women than in elderly men.

Abstract An age-stratified sample of 304 women from Rochester, Minnesota, aged 30–94 years (median 60 years) at baseline underwent measurement of femoral neck bone mineral density (BMD) over a follow-up period extending to 16 years. The average rate of change in femoral neck BMD was −1.0% per year (range −10.0% to +13.4%) and did not vary significantly with age. Because there was no marked increase in the rate of loss around the time of menopause, nor convincing evidence of there being a subset of fast losers, there was fairly good tracking of individual values over time; the correlation of baseline with femoral neck BMD values 16 years later was 0.83. Although a large number of potential determinants was assessed, the only consistent predictor of femoral neck bone loss in women of different ages was baseline femoral neck BMD (r = −0.15; p = 0.023). Otherwise, different sets of risk factors were identified for premenopausal women, women within 20 years of menopause, and women 20 years or more postmenopausal, but the predictive power of these different multivariate models was modest. Nonetheless, these data indicate that femoral neck BMD is quite predictable for extended periods of time. This is reassuring with respect to the use of statistical models that incorporate such data to estimate future fracture risk.

Abstract Although multiple hormones and cytokines regulate various aspects of osteoclast formation, the final two effectors are osteoprotegerin ligand (OPG-L)/osteoclast differentiation factor (ODF), a recently cloned member of the tumor necrosis factor superfamily, and macrophage colony–stimulating factor. OPG-L/ODF is produced by osteoblast lineage cells and exerts its biological effects through binding to its receptor, osteoclast differentiation and activation receptor (ODAR)/receptor activator of NF-κB (RANK), on osteoclast lineage cells, in either a soluble or a membrane-bound form, the latter of which requires cell-to-cell contact. Binding results in rapid differentiation of osteoclast precursors in bone marrow to mature osteoclasts and, at higher concentrations, in increased functional activity and reduced apoptosis of mature osteoclasts. The biological activity of OPG-L/ODF is neutralized by binding to osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF), a member of the TNF-receptor superfamily that also is secreted by osteoblast lineage cells. The biological importance of this system is underscored by the induction in mice of severe osteoporosis by targeted ablation of OPG/OCIF and by the induction of osteopetrosis by targeted ablation of OPG-L/ODF or overexpression of OPG/OCIF. Thus, osteoclast formation may be determined principally by the relative ratio of OPG-L/ODF to OPG/OCIF in the bone marrow microenvironment, and alterations in this ratio may be a major cause of bone loss in many metabolic disorders, including estrogen deficiency and glucocorticoid excess. That changes in but two downstream cytokines mediate the effects of large numbers of upstream hormones and cytokines suggests a regulatory mechanism for osteoclastogenesis of great efficiency and elegance.

Abstract Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2–6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2–6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP-2–stimulated expression of ALP mRNA and protein. The effects of BMP-2 on adipocyte differentiation varied: expression of peroxisome proliferator-activated receptor γ2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP-2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.

Abstract There continues to be uncertainty about the classification of available drugs for treating osteoporosis. We find that grouping them into anti-catabolic and anabolic classes based on the mechanisms of their action on bone remodeling and fracture reduction removes ambiguities and provides a relatively straightforward classification. The recent introduction of teriparatide into clinical practice initiated the era of anabolic therapy for osteoporosis, but it is still unclear how to define an anabolic drug. All drugs that increase bone mass do so by affecting bone remodeling. When their mechanisms of action on bone remodeling and on fracture reduction are considered, we find that anti-osteoporotic drugs fall naturally into either anti-catabolic or anabolic classes. Anti-catabolic drugs increase bone strength and reduce fractures mainly by decreasing the number of bone multicellular units (BMUs). This reduces perforative resorption and preserves skeletal microarchitecture (by preventing further structural damage to trabecular bone and increased porosity in cortical bone induced by high bone remodeling). Reduction in bone remodeling by anti-catabolic drugs may increase bone mass moderately during the interval in which previously initiated BMUs are completing mineralization. Some anti-catabolic drugs may also enhance the formation phase of the remodeling cycle, but their major action is to reduce overall bone turnover (i.e., the number of BMUs in bone). In contrast, anabolic drugs increase bone strength and reduce fractures by substantially increasing bone mass as a result of an overall increase in the number of BMUs combined with a positive BMU balance (the magnitude of the formation phase is greater than that of the resorption phase). Some anabolic drugs also induce renewed modeling, increase periosteal apposition and repair of trabecular microstructure. We hope that this classification will serve as a starting point for continued discussion on the important issue of nomenclature.

Abstract Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312–324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1α,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10−8 M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10−8 M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and β-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.

Abstract We report a 4-year randomized, double-blind, placebo-controlled clinical trial in 236 normal postmenopausal women (mean age ± SE, 66.3 ± 0.2 years) who were randomized to a calcium (1600 mg/day as the citrate) or placebo group. The women were seen every 6 months; 177 completed the trial. Net percentage changes in each group are given relative to baseline. The differences in net percentage changes (calcium group minus placebo group) in medians were: for lumbar spine bone density, 2.0% (p < 0.001) at year 1 and 0.3% (not significant) at year 4; for proximal femur bone density, 1.3% (p = 0.003) at year 1 and 1.3% (p = 0.015) at year 4; and for total body bone mineral, 0.4% (p = 0.002) at year 1 and 0.9% (p = 0.017) at year 4. Similar differences at year 4 were: −18.9% (p = 0.002) for parathyroid hormone (PTH), −11.9% (p = 0.026) for serum osteocalcin, and −32.2% (p = 0.003) for urine free pyridinoline. We conclude that long-term administration of calcium supplements to elderly women partially reverses age-related increases in serum PTH level and bone resorption and decreases bone loss. However, the effects on bone loss were weaker than those reported for estrogen, bisphosphonates, or calcitonin therapy, indicating that calcium supplements alone cannot substitute for these in treating established osteoporosis. Nonetheless, because of their safety, high tolerance, and low expense, calcium supplements may be a useful preventive measure for elderly postmenopausal women whose bone mineral density values are normal for their age.

Abstract We propose here a new unitary model for the pathophysiology of involutional osteoporosis that identifies estrogen (E) deficiency as the cause of both the early, accelerated and the late, slow phases of bone loss in postmenopausal women and as a contributing cause of the continuous phase of bone loss in aging men. The accelerated phase in women is most apparent during the first decade after menopause, involves disproportionate loss of cancellous bone, and is mediated mainly by loss of the direct restraining effects of E on bone cell function. The ensuing slow phase continues throughout life in women, involves proportionate losses of cancellous and cortical bone, and is associated with progressive secondary hyperparathyroidism. This phase is mediated mainly by loss of E action on extraskeletal calcium homeostasis which results in net calcium wasting and increases in the level of dietary calcium intake required to maintain bone balance. Because elderly men have low circulating levels of both bioavailable E and bioavailable testosterone (T) and because recent data suggest that E is at least as important as T in determining bone mass in aging men, E deficiency may also contribute substantially to the continuous bone loss of aging men. In both genders, E deficiency increases bone resorption and may also impair a compensatory increase in bone formation. For the most part, this unitary model is well supported by observational and experimental data and provides plausible explanations to traditional objections to a unitary hypothesis.