Citations

Abstract In a population-based, cross-sectional study, we related age-associated changes in vBMD and in bone structural parameters to circulating bioavailable estradiol and testosterone levels in men. Associations between these bone mass/structural parameters and sex steroid levels were progressively stronger with age. Our previously postulated “threshold” for skeletal estrogen deficiency was most evident at cortical sites. Introduction: Serum sex steroids, particularly estrogen levels, are associated with bone mass in men, and previous work has suggested that there may be a “threshold” bioavailable estradiol (bio E2) level below which the male skeleton becomes estrogen deficient. However, previous studies addressing this issue have exclusively used DXA, which cannot separate trabecular from cortical bone or provide information on bone geometry or structure. Materials and Methods: In an age-stratified population sample of 314 men (age, 22-91 years), we assessed volumetric BMD (vBMD) and bone geometry by QCT at the lumbar spine, femoral neck, distal radius, and distal tibia and related these to circulating bio E2 and bio testosterone (T) levels. Results: Compared with young men (age, 20-39 years), middle-aged men (age, 40-59 years) had significantly lower bio T (−26%, p < 0.001) and bio E2 (−9%, p = 0.038) levels, and these decreases were even greater in the elderly men (age ≥ 60 years, −60% and −38% for bio T and bio E2, respectively, p < 0.001 for both). Reflecting their intact gonadal status, vBMD/structural parameters were not related to sex steroid levels in young men, whereas bio E2 levels were associated consistently with vBMD and variably with bone geometric parameters in the elderly men; middle-aged men showed associations with bio E2 and bio T at some sites. At all cortical sites, vBMD was associated with bio E2 at low (<30 pM, R = 0.27-0.41, p < 0.05-0.001) but not high (≥30 pM, R = −0.003 to 0.12, p = not significant) levels; no such differences were evident at trabecular sites. Conclusions: In men, bio E2 is the most consistent predictor of vBMD and some bone geometric variables as assessed by QCT. We also extend our previous findings on a possible “threshold” for skeletal estrogen deficiency by showing that this is most evident for cortical sites.

Abstract Long-term fracture prediction using bone mineral density remains controversial, as does the additional contribution from assessing bone turnover or clinical risk factors. We measured bone mineral density at various sites, along with biochemical markers of bone turnover, sex steroid levels, and over 100 clinical variables, at baseline on an age-stratified sample of 304 Rochester, MN women in 1980. The 225 postmenopausal women were subsequently followed for 3146 person-years (median, 16.2 years per subject), wherein they experienced 302 new fractures: 81% resulted from minimal or moderate trauma and 60% of these involved the proximal femur, thoracic or lumbar vertebrae, or distal forearm. Accounting for multiple fractures per subject, these osteoporotic fractures together were best predicted by baseline femoral neck bone mineral density (age-adjusted hazard ratio [HR] per SD decrease, 1.37; 95% CI, 1.10–1.70); 19 moderate trauma forearm fractures were best predicted by distal radius bone mineral content, whereas 28 hip fractures and 100 vertebral fractures were best predicted by femoral neck bone mineral density. Femoral neck bone mineral density performed comparably in predicting osteoporotic fracture risk within the first decade of follow-up (HR, 1.38; 95% CI, 1.10–1.74) as well as more than 10 years after baseline (HR, 1.39; 95% CI, 1.05–1.84). The older biochemical markers were not associated with fractures, but serum “free” estradiol index was independently predictive of short- and long-term fracture risk. Consistent clinical risk factors were not identified, but statistical power was limited. Identifying patients at increased long-term risk of fracture is challenging, but it is reassuring that femoral neck bone mineral density can predict osteoporotic fractures up to 20 years later.

Abstract Runx2/Cbfa1 has been identified as a “master gene” controlling osteoblast differentiation. However, its role in inducing the osteoblast phenotype has been characterized primarily in rodent systems. Thus, we examined Runx2/Cbfa1 messenger RNA, protein, and activity levels during osteoblastic differentiation of human bone marrow stromal (BMSC) cells. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the expression of alkaline phosphatase and osteocalcin mRNAs increased in a time-dependent manner with the development of the osteoblast phenotype by these cells (hMS2–15). Type II Runx2/Cbfa1 messenger RNA was found to be constitutively expressed in hMS2–15 cells and not altered during differentiation; there was no detectable expression of the type I Runx2/Cbfa1 transcript. Interestingly, despite the absence of any change in Runx2/Cbfa1 messenger RNA levels during osteoblastic differentiation of these cells, the activity of Runx2/Cbfa1, as assessed by binding to the osteoblast-specific cis-acting element 2 (OSE2), increased markedly at all time-points examined, with the highest activity level seen at day 7. Similar results were observed in primary cultures of less differentiated human marrow-derived mesenchymal stem cells. Immunoprecipitation and Western blot analysis revealed that whereas there was no increase in Runx2/Cbfa1 protein levels with differentiation in hMS2–15 cells, there was an increase in Runx2/Cbfa1 phosphorylation. Thus, in contrast to rodent systems where osteoblast differentiation is associated with increased synthesis of Runx2/Cbfa1, we find that in human BMSC, osteoblastic differentiation is associated primarily with increases in Runx2/Cbfa1 activity, without a change in messenger RNA or protein levels. Our findings also show that the increase in Runx2/Cbfa1 activity occurs through a posttranslational mechanism involving phosphorylation of key residues.

Abstract Estrogen induction of progesterone receptor (PR) expression may be important to bone physiology because progesterone has been implicated in the control of bone formation and resorption. Although PR gene expression can be induced in osteoblasts by estrogen signaling through the estrogen receptor (ER) α isoform, it is unknown whether the ER-β isoform is involved in this regulation. The effect of estrogen on PR expression was examined in human fetal osteoblast (hFOB) cell lines stably transfected with either ER-α or ER-β. Estrogen treatment of hFOB/ER-α cells induced PR messenger RNA (mRNA) steady-state levels after 24 h and protein levels after 48 h, as established by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Interestingly, no induction of PR expression was observed in the hFOB/ER-β cells during this period. However, PR mRNA was induced progressively after 48 h of treatment with estrogen with maximum levels achieved at 12 days posttreatment. ER protein also was increased after 12 days of treatment. Both A and B isoforms of PR (PRA and PRB) were induced by estrogen in the hFOB/ER-α cells as well as much later in hFOB/ER-β cells. The pure antiestrogen ICI 182,780 prevented PR induction by estrogen in both cell lines. An ER-β-selective antagonist R, R-tetrahydrochrysene (THC) abolished the induction of PR mRNA in hFOB/ER-β but not in hFOB/ER-α cells, verifying that the response in the former cell line was ER-β-mediated. Transient cotransfection of hFOB cells with ER-α or ER-β together with either a human PRA or PRB promoter linked to a reporter plasmid revealed that although the PRB promoter was stimulated equally by estrogen activation of either ER isoform, PRA was activated preferentially by ER-α. Together, these results show that although estrogen can up-regulate endogenous PR gene expression in osteoblasts via both ER isoforms, ER-α is the predominant inducer.

Abstract Although median levels of bone turnover are increased in postmenopausal women, it is unclear whether the low circulating levels of endogenous estrogen exert a regulatory role on these levels. This issue was evaluated by assessing the effect of a blockade of estrogen synthesis on bone turnover markers in 42 normal women (mean age ± SD, 69 ± 5 years) randomly assigned to groups receiving the potent aromatase inhibitor letrozole or placebo for 6 months. Letrozole treatment reduced serum estrone (E1) and estradiol (E2) to near undetectable levels (p < 0.0001). This treatment did not affect bone formation markers but, as compared with the placebo group, increased bone resorption markers (urine 24-h pyridinoline [PYD] by 13.3% [p < 0.05] and 24-h urine deoxypyridinoline [DPD] by 14.2% [p < 0.05]) and decreased serum parathyroid hormone (PTH) by 22% (p = 0.002). These data indicate that in late postmenopausal women even the low serum estrogen levels present exert a restraining effect on bone turnover and support the concept that variations in these low levels may contribute to differences in their rate of bone loss.

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.