The endogenous selective estrogen receptor modulator 27-hydroxycholesterol is a negative regulator of bone homeostasis

Abstract

Osteoporosis is an important clinical problem, affecting more than 50% of people over age 50 yr. Estrogen signaling is critical for maintaining proper bone density, and the identification of an endogenous selective estrogen receptor (ER) modulator, 27-hydroxycholesterol (27HC), suggests a mechanism by which nutritional/metabolic status can influence bone biology. With its levels directly correlated with cholesterol, a new possibility emerges wherein 27HC links estrogen and cholesterol signaling to bone homeostasis. In these studies, we found that increasing concentrations of 27HC, both by genetic and pharmacological means, led to decreased bone mineral density that was associated with decreased bone formation and increased bone resorption. Upon manipulation of endogenous estrogen levels, many of the responses to elevated 27HC were altered in such a way as to implicate ER as a likely mediator. In a model of postmenopausal bone loss, some pathologies associated with elevated 27HC were exacerbated by the absence of endogenous estrogens, suggesting that 27HC may act both in concert with and independently from classic ER signaling. These data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis. Patients with high cholesterol likely also have higher than average 27HC, perhaps putting them at a higher risk for bone loss and fracture. More studies are warranted to fully elucidate the mechanism of action of 27HC in bone and to identify ways to modulate this pathway therapeutically.

Figure 1

Altered trabecular bone in the absence of 27HC. The lumbar spine and femur from female mice at 10 wk of age were harvested for analysis. A, There was no significant difference in BMD between the Cyp27a1^+/+ and Cyp27a1^−/− mice. B, Trabecular (Trab.) morphometry was assessed by qCT on the femur. BV/TV, Bone volume/total volume. Data (n = 7–8) are shown as the mean ± sem. **, P < 0.01 by unpaired two-tailed t test on raw data.

Figure 2

Pathological elevation of 27HC decreases trabecular bone density. The lumbar spine and femur from female mice at 10 wk of age were harvested for analysis. A, The BMD in the lumbar spine was significantly lower in Cyp7b1^−/− compared with wild-type mice (unpaired two-tailed t test on raw data, P < 0.001). B, Trabecular (Trab.) morphometry was assessed by qCT on the femur. BV/TV, Bone volume/total volume. For A and B, data (n = 9–10) are shown as the mean ± sem. ***, P < 0.001; **, P < 0.01; *, P < 0.05 by unpaired two-tailed t test on raw data. C, qCT image of the spine from representative Cyp7b1^−/− and Cyp7b1^+/+ intact female mice. The 8-μm slice was decided at the same point via grossly determining the same bone epicondyle. D, H&E staining of the proximal tibia from representative Cyp7b1^−/− and Cyp7b1^+/+ intact female mice.

Figure 3

27HC supplementation decreases bone parameters. Cyp7b1^+/+ mice were treated with placebo or 27HC for 28 d before harvesting the lumbar spine, femur, and calvaria. A, Bones were subjected to DEXA analysis. Data (n = 6–8) are shown as the mean ± sem. *, P < 0.05 by unpaired two-tailed t test on raw data. B, Trabecular (Trab.) morphometry was assessed by qCT on the femur. BV/TV, Bone volume/total volume. Data (n = 5–8) are shown as the mean ± sem. *, P < 0.05; ^, P = 0.0506 by unpaired two-tailed t test on raw data. C, Gene expression was analyzed in the isolated calvaria. The expression for OPG, alkaline phosphatase, osteocalcin, and RANKL was quantified by qPCR and normalized to cyclophilin. Data (n = 3–5) are presented with respect to placebo (mean ± sem). *, P < 0.05 compared with placebo (unpaired t test).

Figure 4

Estrogen attenuates the negative impact of 27HC on the bone. A, Cyp7b1^−/− mice were treated with placebo or E2 (10 or 20 μg/kg) for 28 d. Data (n = 4–5) are shown as the mean ± sem. ***, P < 0.001; *, P < 0.05 compared with the placebo group (unpaired two-tailed t test on raw data). B, Trabecular (Trab.) morphometry was assessed by qCT on the femur. Mice were treated with placebo or 20 μg/kg E2 for 28 d. BV/TV, Bone volume/total volume. Data (n = 4–5) are presented as the mean ± sem. ***, P < 0.001; **, P < 0.01 compared with the placebo group (one-way ANOVA followed by Student-Newman-Keuls).

Figure 5

A model of postmenopausal osteoporosis is exacerbated by high levels of 27HC. The lumbar spine and femur were harvested for analysis. A, Mice (6 wk old) were assigned to sham surgery or OVX with placebo (OVX P) or E2 (OVX E) treatment for 28 d. Data (n = 9–10) are shown as the mean ± sem. B, Trabecular (Trab.) morphometry was assessed by qCT on the femur. BV/TV, Bone volume/total volume. Data (n = 9–10) are shown as the mean ± sem. Mice were assigned to sham surgery or OVX with placebo (OVX P) or E2 (OVX E) treatment for 28 d. Different letters denote statistical significance (one-way ANOVA followed by Student-Newman-Keuls, P < 0.05).

Figure 6

Deregulated OB and OC function upon increasing 27HC. A, Urine was collected and analyzed for DPD cross-links. *, P < 0.05 compared with the sham-operated Cyp7b1^+/+ group (one-way ANOVA followed by Student-Newman-Keuls); ***, P < 0.001. B, Serum was collected and analyzed for osteocalcin by ELISA. ^, P < 0.05 compared with the sham-operated Cyp7b1^−/− group (one-way ANOVA followed by Student-Newman-Keuls). C, Lumbar spine was analyzed by histomorphometry. **, P < 0.01 by unpaired two-tailed t test on raw data.

Figure 7

27HC acts as a partial ER agonist in primary calvarial pre-OBs. A, The effect of 27HC alone or in combination with E2 on alkaline phosphatase (Alk. Phos.) activity was assessed at the indicated dose. B, The effect of E2 (10 nm) and 27HC (1 μm) on alkaline phosphatase (Alk. Phos.) activity is blocked by the pure antiestrogen (1 μm ICI 182,780). Calvarial pre-OBs were treated for 36 h with vehicle or the indicated ligand, after which time alkaline phosphatase activity was determined. Data (n = 6) are expressed with respect to total protein (mean ± sem). Different letters denote statistical significance (one-way ANOVA followed by Student-Newman-Keuls, P < 0.05).