Louise K. McCready

Relation of age, gender, and bone mass to circulating sclerostin levels in women and men.

Sclerostin is a potent inhibitor of Wnt signaling and bone formation. However, there is currently no information on the relation of circulating sclerostin levels to age, gender, or bone mass in humans. Thus we measured serum sclerostin levels in a population‐based sample of 362 women [123 premenopausal, 152 postmenopausal not on estrogen treatment (ET), and 87 postmenopausal on ET] and 318 men, aged 21 to 97 years. Sclerostin levels (mean ± SEM) were significantly higher in men than women (33.3 ± 1.0 pmol/L versus 23.7 ± 0.6 pmol/L, p < .001). In pre‐ and postmenopausal women not on ET combined (n = 275) as well as in men, sclerostin levels were positively associated with age (r = 0.52 and r = 0.64, respectively, p < .001 for both). Over life, serum sclerostin levels increased by 2.4‐ and 4.6‐fold in the women and men, respectively. Moreover, for a given total‐body bone mineral content, elderly subjects (age ≥ 60 years) had higher serum sclerostin levels than younger subjects (ages 20 to 39 years). Our data thus demonstrate that (1) men have higher serum sclerostin levels than women, (2) serum sclerostin levels increase markedly with age, and (3) compared with younger subjects, elderly individuals have higher serum sclerostin levels for a given amount of bone mass. Further studies are needed to define the cause of the age‐related increase in serum sclerostin levels in humans as well as the potential role of this increase in mediating the known age‐related impairment in bone formation.

In vivo assessment of bone quality in postmenopausal women with type 2 diabetes

Although patients with type 2 diabetes (T2D) are at significant risk for well‐recognized diabetic complications, including macrovascular disease, retinopathy, nephropathy, and neuropathy, it is also clear that T2D patients are at increased risk for fragility fractures. Furthermore, fragility fractures in patients with T2D occur at higher bone mineral density (BMD) values compared to nondiabetic controls, suggesting abnormalities in bone material strength (BMS) and/or bone microarchitecture (bone “quality”). Thus, we performed in vivo microindentation testing of the tibia to directly measure BMS in 60 postmenopausal women (age range, 50–80 years) including 30 patients diagnosed with T2D for >10 years and 30 age‐matched, nondiabetic controls. Regional BMD was measured by dual‐energy X‐ray absorptiometry (DXA); cortical and trabecular bone microarchitecture was assessed from high‐resolution peripheral quantitative computed tomography (HRpQCT) images of the distal radius and tibia. Compared to controls, T2D patients had significantly lower BMS: unadjusted (−11.7%; p < 0.001); following adjustment for body mass index (BMI) (−10.5%; p < 0.001); and following additional adjustment for age, hypertension, nephropathy, neuropathy, retinopathy, and vascular disease (−9.2%; p = 0.022). By contrast, after adjustment for confounding by BMI, T2D patients had bone microarchitecture and BMD that were not significantly different than controls; however, radial cortical porosity tended to be higher in the T2D patients. In addition, patients with T2D had significantly reduced serum markers of bone turnover (all p < 0.001) compared to controls. Of note, in patients with T2D, the average glycated hemoglobin level over the previous 10 years was negatively correlated with BMS (r = −0.41; p = 0.026). In conclusion, these findings represent the first demonstration of compromised BMS in patients with T2D. Furthermore, our results confirm previous studies demonstrating low bone turnover in patients with T2D and highlight the potential detrimental effects of prolonged hyperglycemia on bone quality. Thus, the skeleton needs to be recognized as another important target tissue subject to diabetic complications.

Effects of Parathyroid Hormone Treatment on Circulating Sclerostin Levels in Postmenopausal Women

CONTEXT Intermittent PTH treatment stimulates bone formation, but the mechanism(s) of this effect remain unclear. Sclerostin is an inhibitor of Wnt signaling, and animal studies have demonstrated that PTH suppresses sclerostin production. OBJECTIVE The objective of the study was to test whether intermittent PTH treatment of postmenopausal women alters circulating sclerostin levels. DESIGN Prospective study. SETTING The study was conducted at a clinical research unit. PARTICIPANTS AND INTERVENTIONS Participants included 27 postmenopausal women treated with PTH (1-34) for 14 d and 28 control women. MAIN OUTCOME MEASURES Serum sclerostin levels were measured. RESULTS Circulating sclerostin levels decreased significantly in the PTH-treated subjects, from (mean ± SEM) 551 ± 32 to 482 ± 31 pg/ml (-12.7%, P < 0.0001) but did not change in the control women (baseline, 559 ± 34 pg/ml; end point, 537 ± 40 pg/ml, P = 0.207; P = 0.017 for difference in changes between groups). Bone marrow plasma was obtained in a subset of the control and PTH-treated subjects (n = 19 each) at the end of the treatment period, and marrow plasma and peripheral serum sclerostin levels were significantly correlated (R = 0.64, P < 0.0001). Marrow plasma sclerostin levels were 24% lower in PTH-treated compared with control women, but perhaps due to the smaller sample size, this difference was not statistically significant (P = 0.173). CONCLUSIONS Circulating sclerostin levels correlate with bone marrow plasma levels and are reduced by intermittent PTH therapy in postmenopausal women. Further studies are needed to assess the extent to which decreases in sclerostin production contribute to the anabolic skeletal response to PTH.

Bone Structure at the Distal Radius During Adolescent Growth

The incidence of distal forearm fractures peaks during the adolescent growth spurt, but the structural basis for this is unclear. Thus, we studied healthy 6‐ to 21‐yr‐old girls (n = 66) and boys (n = 61) using high‐resolution pQCT (voxel size, 82 μm) at the distal radius. Subjects were classified into five groups by bone‐age: group I (prepuberty, 6–8 yr), group II (early puberty, 9–11 yr), group III (midpuberty, 12–14 yr), group IV (late puberty, 15–17 yr), and group V (postpuberty, 18–21 yr). Compared with group I, trabecular parameters (bone volume fraction, trabecular number, and thickness) did not change in girls but increased in boys from late puberty onward. Cortical thickness and density decreased from pre‐ to midpuberty in girls but were unchanged in boys, before rising to higher levels at the end of puberty in both sexes. Total bone strength, assessed using microfinite element models, increased linearly across bone age groups in both sexes, with boys showing greater bone strength than girls after midpuberty. The proportion of load borne by cortical bone, and the ratio of cortical to trabecular bone volume, decreased transiently during mid‐ to late puberty in both sexes, with apparent cortical porosity peaking during this time. This mirrors the incidence of distal forearm fractures in prior studies. We conclude that regional deficits in cortical bone may underlie the adolescent peak in forearm fractures. Whether these deficits are more severe in children who sustain forearm fractures or persist into later life warrants further study.

Regulation of circulating sclerostin levels by sex steroids in women and in men

Sex steroids are important regulators of bone turnover, but the mechanisms of their effects on bone remain unclear. Sclerostin is an inhibitor of Wnt signaling, and circulating estrogen (E) levels are inversely associated with sclerostin levels in postmenopausal women. To directly test for sex steroid regulation of sclerostin levels, we examined effects of E treatment of postmenopausal women or selective withdrawal of E versus testosterone (T) in elderly men on circulating sclerostin levels. E treatment of postmenopausal women (n = 17) for 4 weeks led to a 27% decrease in serum sclerostin levels [versus +1% in controls (n = 18), p < .001]. Similarly, in 59 elderly men, we eliminated endogenous E and T production and studied them under conditions of physiologic T and E replacement, and then following withdrawal of T or E, we found that E, but not T, prevented increases in sclerostin levels following induction of sex steroid deficiency. In both sexes, changes in sclerostin levels correlated with changes in bone‐resorption, but not bone‐formation, markers (r = 0.62, p < .001, and r = 0.33, p = .009, for correlations with changes in serum C‐terminal telopeptide of type 1 collagen in the women and men, respectively). Our studies thus establish that in humans, circulating sclerostin levels are reduced by E but not by T. Moreover, consistent with recent data indicating important effects of Wnts on osteoclastic cells, our findings suggest that in humans, changes in sclerostin production may contribute to effects of E on bone resorption.

Effect of Blockade of TNF-α and Interleukin-1 Action on Bone Resorption in Early Postmenopausal Women†‡

After acute estrogen withdrawal in postmenopausal women, administration of anakinra or etanercept, specific blockers of IL‐1 and TNF‐α, respectively, reduced the rise in bone resorption markers to about one half of that in controls. This is consistent with an important role for these immune cytokines in mediating the effect of estrogen deficiency on bone.

Effects of Estrogen on Osteoprogenitor Cells and Cytokines/Bone-Regulatory Factors in Postmenopausal Women

Decreases in estrogen levels contribute not only to early postmenopausal bone loss but also to bone loss with aging. While estrogen is critical for the maintenance of bone formation, the mechanism(s) of this effect remain unclear. Thus, we assessed the effects of 4months of transdermal estradiol treatment (0.05mg/day) of postmenopausal women as compared to no treatment (n=16 per group) on the expression of genes in pre-specified pathways in freshly isolated bone marrow osteoprogenitor cells (hematopoietic lineage [lin]-/Stro1+). We also evaluated whether estrogen treatment modulated peripheral blood or bone marrow plasma levels of the Wnt antagonists, sclerostin and DKK1, as well as serotonin, OPG, RANKL, adiponectin, oxytocin, and inflammatory cytokines (TNFα, IL-1β, and IL-6), as each of these molecules have recently been shown to play an important role in regulating osteoblast function and/or being responsive to estrogen. We observed a significant decrease in the expression of several proliferation markers (cyclin B1, cyclin E1, E2F1) and increase in adhesion molecules (N-cadherin) in bone marrow lin-/Stro1+ cells from estrogen-treated compared to control women. None of the peripheral blood or bone marrow plasma marker levels differed between the two groups, with the exception of sclerostin levels, which were significantly lower in the estrogen-treated as compared to the control women in peripheral serum (by 32%, P=0.009) and in bone marrow plasma (by 34%, P=0.017). There were significant differences in bone marrow versus peripheral plasma levels of several factors: sclerostin and OPG levels were higher in bone marrow as compared to peripheral plasma, whereas serotonin and adiponectin levels were higher in peripheral as compared to bone marrow plasma. In summary, our data directly assessing possible regulation by estrogen of osteoprogenitor cells in humans indicate that, consistent with previous studies in mice, estrogen suppresses the proliferation of human bone marrow lin-/Stro1+ cells, which likely represent early osteoprogenitor cells. Further animal and human studies are needed to define the role of the changes we observed in mRNAs for adhesion molecules in these cells and in local sclerostin production in bone in mediating the effects of estrogen on bone metabolism in humans.

Effects of Suppression of Follicle-Stimulating Hormone Secretion on Bone Resorption Markers in Postmenopausal Women

CONTEXT It has recently been proposed that the increase in bone resorption after the menopause may not be due principally to estrogen deficiency but rather to the concomitant increase in circulating FSH levels. OBJECTIVE The objective of the study was to test whether suppression of FSH secretion in postmenopausal women reduces levels of bone resorption markers. DESIGN This was a prospective study. SETTING The study was conducted at a clinical research unit. PARTICIPANTS AND INTERVENTIONS Postmenopausal women were treated with a GnRH agonist (leuprolide acetate, 7.5 mg im every 28 d; n = 21) or placebo injections (control; n = 20). Both groups received the aromatase inhibitor, letrozole, 2.5 mg/d, to eliminate variations in endogenous estrogen levels as a confounder. MAIN OUTCOME MEASURES Serum FSH and bone resorption markers [serum C-terminal telopeptide of type I collagen (CTX) and tartrate-resistant acid phosphatase 5b (TRAP5b)] at d 105 (3.5 months) of treatment as compared with baseline. RESULTS Compared with baseline, serum FSH levels did not change significantly in controls (+6%) but were reduced (-86%, into the premenopausal range) in the GnRH group. Due to the aromatase inhibitor-induced reduction in estrogen production, serum CTX and TRAP5b levels increased significantly in controls (+20 and +10%, respectively). In the GnRH group, suppression of FSH secretion did not reduce serum CTX or TRAP5b levels; rather, both markers also increased in these women (+34 and +15%, respectively; P = 0.161 and 0.266 for comparison of percent changes between groups). CONCLUSIONS This direct interventional study demonstrates that FSH does not regulate bone resorption in postmenopausal women.

Sclerostin levels during growth in children

SummarySerum sclerostin levels are associated with cortical porosity, suggesting that changes in sclerostin production during growth may play a role in defining cortical structure.IntroductionSclerostin, produced by osteocytes, is a potent inhibitor of Wnt signaling and bone formation. While sclerostin levels increase with age in adults and are higher in men compared to women, there is currently no information on changes in circulating sclerostin levels during growth in humans.MethodsWe measured serum sclerostin levels in 6- to 21-year-old girls (n = 62) and boys (n = 56) and related these to trabecular and cortical bone microarchitectural parameters using high-resolution peripheral quantitative computed tomography and to markers of bone turnover.ResultsSerum sclerostin levels were higher in boys as compared to girls and declined in both sexes following the onset of puberty. There was no consistent relationship between sclerostin levels and trabecular bone parameters in either sex. However, serum sclerostin levels were inversely associated with cortical volumetric bone mineral density and cortical thickness in girls and positively associated with the cortical porosity index in both girls and boys. Bone turnover markers were positively correlated with serum sclerostin levels in both sexes.ConclusionThe gender difference in serum sclerostin levels appears to be established during puberty, and sclerostin levels tend to decline in late puberty in both girls and boys. Serum sclerostin levels are associated with cortical porosity, suggesting that changes in sclerostin production during growth may play a role in defining cortical structure.

Effects of age on bone mRNA levels of sclerostin and other genes relevant to bone metabolism in humans

Although aging is associated with a decline in bone formation in humans, the molecular pathways contributing to this decline remain unclear. Several previous clinical studies have shown that circulating sclerostin levels increase with age, raising the possibility that increased production of sclerostin by osteocytes leads to the age-related impairment in bone formation. Thus, in the present study, we examined circulating sclerostin levels as well as bone mRNA levels of sclerostin using quantitative polymerase chain reaction (QPCR) analyses in needle bone biopsies from young (mean age, 30.0years) versus old (mean age, 72.9years) women. In addition, we analyzed the expression of genes in a number of pathways known to be altered with skeletal aging, based largely on studies in mice. While serum sclerostin levels were 46% higher (p<0.01) in the old as compared to the young women, bone sclerostin mRNA levels were no different between the two groups (p=0.845). However, genes related to notch signaling were significantly upregulated (p=0.003 when analyzed as a group) in the biopsies from the old women. In an additional analysis of 118 genes including those from genome-wide association studies related to bone density and/or fracture, BMP/TGFβ family genes, selected growth factors and nuclear receptors, and Wnt/Wnt-related genes, we found that mRNA levels of the Wnt inhibitor, SFRP1, were significantly increased (by 1.6-fold, p=0.0004, false discovery rate [q]=0.04) in the biopsies from the old as compared to the young women. Our findings thus indicate that despite increases in circulating sclerostin levels, bone sclerostin mRNA levels do not increase in elderly women. However, aging is associated with alterations in several key pathways and genes in humans that may contribute to the observed impairment in bone formation. These include notch signaling, which represents a potential therapeutic target for increasing bone formation in humans. Our studies further identified mRNA levels of SFRP1 as being increased in aging bone in humans, suggesting that this may also represent a viable target for the development of anabolic therapies for age-related bone loss and osteoporosis.