Sofia Movérare

Differential effects on bone of estrogen receptor α and androgen receptor activation in orchidectomized adult male mice

Androgens may regulate the male skeleton either directly by stimulation of the androgen receptor (AR) or indirectly by aromatization of androgens into estrogens and, thereafter, by stimulation of the estrogen receptors (ERs). To directly compare the effect of ER activation on bone in vivo with the effect of AR activation, 9-month-old orchidectomized wild-type and ER-inactivated mice were treated with the nonaromatizable androgen 5α-dihydrotestosterone, 17β-estradiol, or vehicle. Both ERα and AR but not ERβ activation preserved the amount of trabecular bone. ERα activation resulted both in a preserved thickness and number of trabeculae. In contrast, AR activation exclusively preserved the number of trabeculae, whereas the thickness of the trabeculae was unaffected. Furthermore, the effects of 17β-estradiol could not be mediated by the AR, and the effects of 5α-dihydrotestosterone were increased rather than decreased in ER-inactivated mice. ERα, but not AR or ERβ, activation resulted in preserved thickness, volumetric density, and mechanical strength of the cortical bone. ERα activation increased serum levels of insulin-like growth factor I, which were positively correlated with all the cortical and trabecular bone parameters that were specifically preserved by ERα activation but not by AR activation, suggesting that insulin-like growth factor I might mediate these effects of ERα activation. Thus, the in vivo bone-sparing effect of ERα activation is distinct from the bone-sparing effect of AR activation in adult male mice. Because these two pathways are clearly distinct from each other, one may speculate that a combined treatment of selective ER modulators and selective AR modulators might be beneficial in the treatment of osteoporosis.

Effects of liver-derived insulin-like growth factor I on bone metabolism in mice

Insulin‐like growth factor (IGF) I is an important regulator of both skeletal growth and adult bone metabolism. To better understand the relative importance of systemic IGF‐I versus locally expressed IGF‐I we have developed a transgenic mouse model with inducible specific IGF‐I gene inactivation in the liver (LI‐IGF‐I−/−). These mice are growing normally up to 12 weeks of age but have a disturbed carbohydrate and lipid metabolism. In this study, the long‐term effects of liver‐specific IGF‐I inactivation on skeletal growth and adult bone metabolism were investigated. The adult (week 8–55) axial skeletal growth was decreased by 24% in the LI‐IGF‐I−/− mice whereas no major reduction of the adult appendicular skeletal growth was seen. The cortical cross‐sectional bone area, as measured in the middiaphyseal region of the long bones, was decreased in old LI‐IGF‐I−/− mice. This reduction in the amount of cortical bone was caused mainly by decreased periosteal circumference and was associated with a weaker bone determined by a decrease in ultimate load. In contrast, the amount of trabecular bone was not decreased in the LI‐IGF‐I−/− mice. DNA microarray analysis of 30‐week‐old LI‐IGF‐I−/− and control mice indicated that only four genes were regulated in bone whereas ∼40 genes were regulated in the liver, supporting the hypothesis that liver‐derived IGF‐I is of minor importance for adult bone metabolism. In summary, liver‐derived IGF‐I exerts a small but significant effect on cortical periosteal bone growth and on adult axial skeletal growth while it is not required for the maintenance of the trabecular bone in adult mice.

Estrogen Receptor-β Inhibits Skeletal Growth and Has the Capacity to Mediate Growth Plate Fusion in Female Mice†

To determine the long‐term role of ERβ in the regulation of longitudinal bone growth, appendicular and axial skeletal growth was followed and compared in female ERβ−/−, ERα−/−, and ERα−/−β−/− mice. Our results show that ERβ inhibits appendicular and axial skeletal growth and has the capacity to induce fusion of the growth plates.

Two Different Pathways for the Maintenance of Trabecular Bone in Adult Male Mice

Androgens may regulate the male skeleton either directly via activation of the androgen receptor (AR) or indirectly via aromatization of androgens into estrogen and, thereafter, via activation of estrogen receptors (ERs). There are two known estrogen receptors, ER‐α and ER‐β. The aim of this study was to investigate the relative roles of ER‐α, ER‐β, and AR in the maintenance of trabecular bone in male mice. Seven‐month‐old male mice, lacking ER‐α (ERKO), ER‐β (BERKO), or both receptors (DERKO), were orchidectomized (orx) and treated for 3 weeks with 0.7 μg/mouse per day of 17β‐estradiol or vehicle. No reduction in trabecular bone mineral density (BMD) was seen in ERKO, BERKO, or DERKO mice before orx, showing that neither ER‐α nor ER‐β is required for the maintenance of a normal trabecular BMD in male mice. After orx, there was a pronounced decrease in trabecular BMD, similar for all groups, resulting in equal levels of trabecular BMD in all genotypes. This reduction was reversed completely in wild‐type (WT) and BERKO mice treated with estrogen, and no significant effect of estrogen was found in ERKO or DERKO mice. In summary, the trabecular bone is preserved both by a testicular factor, presumably testosterone acting via AR and by an estrogen‐induced activation of ER‐α. These results indicate that AR and ER‐α are redundant in the maintenance of the trabecular bone in male mice. In contrast, ER‐β is of no importance for the regulation of trabecular bone in male mice.

Identification of Estrogen-Regulated Genes of Potential Importance for the Regulation of Trabecular Bone Mineral Density†

Estrogen is of importance for the regulation of trabecular bone mineral density (BMD). The aim of this study was to search for possible mechanisms of action of estrogen on bone. Ovariectomized (OVX) mice were treated with 17β‐estradiol. Possible effects of estrogen on the expression of 125 different bone‐related genes in humerus were analyzed using the microarray technique. Estrogen regulated 12 of these genes, namely, two growth factor‐related genes, 8 cytokines, and 2 bone matrix‐related genes. Five of the 12 genes are known to be estrogen‐regulated, and the remaining 7 genes are novel estrogen‐regulated genes. Seven genes, including interleukin‐1 receptor antagonist (IL‐1ra), IL‐1receptor type II (IL‐1RII), insulin‐like growth factor‐binding protein 4 (IGFBP‐4), transforming growth factor β (TGF‐β), granulocyte colony‐stimulating factor receptor (G‐CSFR), leukemia inhibitory factor receptor (LIFR), and soluble IL‐4 receptor (sIL‐4R) were selected as probable candidate genes for the trabecular bone‐sparing effect of estrogen, as the mRNA levels of these genes were highly correlated (r2 > 0.65) to the trabecular BMD. The regulation of most of these seven genes was predominantly estrogen receptor α (ER‐α)‐mediated (5/7) while some genes (2/7) were regulated both via ER‐α and ER‐β. In conclusion, by using the microarray technique, we have identified four previously known and three novel estrogen‐regulated genes of potential importance for the trabecular bone‐sparing effect of estrogen.

Estren-mediated inhibition of T lymphopoiesis is estrogen receptor-independent whereas its suppression of T cell-mediated inflammation is estrogen receptor-dependent

Estrogen has extensive effects on the immune system. The aim of the present experiments was to compare the effects of 17β‐estradiol (E2) and 4‐estren‐3α,17β‐diol (estren) on T lymphopoiesis and T cell‐dependent inflammation. In order to investigate the role of estrogen receptors (ER) in the effects of E2 and estren on the immune system, ER knock‐out mice lacking both ERα and ERβ (DERKO) were used. T lymphopoiesis and T cell‐dependent inflammation were studied by investigating thymus cellularity, the delayed‐type hypersensitivity (DTH) reaction, CD4+ T cells in spleen and serum levels of interleukin (IL)‐6. As expected, the presence of ERs was mandatory for all the effects of E2. In contrast, treatment with estren reduced thymus cellularity in ER knock‐out mice, indicating an effect through ER‐independent pathways. Interestingly, estren suppressed only DTH, the frequency of CD4+ T cells in spleen and serum levels of IL‐6 in wild‐type (WT) mice, but not in mice lacking ERs. Thus, our study is the first to show that estren inhibits T lymphopoiesis via ER‐independent pathways, whereas its suppressive effects on inflammation are ER‐dependent.