Androgens and bone.

Loss of estrogens or androgens increases the rate of bone remodeling by removing restraining effects on osteoblastogenesis and osteoclastogenesis, and also causes a focal imbalance between resorption and formation by prolonging the lifespan of osteoclasts and shortening the lifespan of osteoblasts. Conversely, androgens, as well as estrogens, maintain cancellous bone mass and integrity, regardless of age or sex. Although androgens, via the androgen receptor (AR), and estrogens, via the estrogen receptors (ERs), can exert these effects, their relative contribution remains uncertain. Recent studies suggest that androgen action on cancellous bone depends on (local) aromatization of androgens into estrogens. However, at least in rodents, androgen action on cancellous bone can be directly mediated via AR activation, even in the absence of ERs. Androgens also increase cortical bone size via stimulation of both longitudinal and radial growth. First, androgens, like estrogens, have a biphasic effect on endochondral bone formation: at the start of puberty, sex steroids stimulate endochondral bone formation, whereas they induce epiphyseal closure at the end of puberty. Androgen action on the growth plate is, however, clearly mediated via aromatization in estrogens and interaction with ERalpha. Androgens increase radial growth, whereas estrogens decrease periosteal bone formation. This effect of androgens may be important because bone strength in males seems to be determined by relatively higher periosteal bone formation and, therefore, greater bone dimensions, relative to muscle mass at older age. Experiments in mice again suggest that both the AR and ERalpha pathways are involved in androgen action on radial bone growth. ERbeta may mediate growth-limiting effects of estrogens in the female but does not seem to be involved in the regulation of bone size in males. In conclusion, androgens may protect men against osteoporosis via maintenance of cancellous bone mass and expansion of cortical bone. Such androgen action on bone is mediated by the AR and ERalpha.

Increased adipogenesis in bone marrow but decreased bone mineral density in mice devoid of thyroid hormone receptors.

Mice deficient for all known thyroid hormone receptors, TRalpha1-/-beta-/- mice, display a clear skeletal phenotype characterized by growth retardation, delayed maturation of long bones and decreased trabecular and total bone mineral density (BMD; -14.6 +/- 2.8%, -14.4 +/- 1.5%). The aim of the present study was to investigate the molecular mechanisms behind the skeletal phenotype in TRalpha1-/-beta-/- mice. Global gene expression analysis was performed on total vertebrae from wild-type (WT) and TRalpha1-/-beta-/- mice using DNA microarray and the results were verified by real-time PCR. The mRNA levels of six genes (AdipoQ, Adipsin, Fat-Specific Protein 27 (FSP 27), lipoprotein lipase (LPL), retinol-binding protein (RBP) and phosphoenolpyruvate carboxykinase (PEPCK)) expressed by mature adipocytes were increased in TRalpha1-/-beta-/- compared with WT mice. An increased amount of fat (225% over WT) due to an increased number but unchanged mean size of adipocytes in the bone marrow of TRalpha1-/-beta-/- mice was revealed. Interestingly, the mRNA levels of the key regulator of osteoclastogenesis, receptor activator of NF-varkappab ligand (RANKL), were dramatically decreased in TRalpha1-/-beta-/- mice. In conclusion, TRalpha1-/-beta-/- mice demonstrated increased expression of adipocyte specific genes and an increased amount of bone marrow fat. Thus, these mice have increased adipogenesis in bone marrow associated with decreased trabecular bone mineral density (BMD). One may speculate that these effects either could be caused by an imbalance in the differentiation of the osteoblast and the adipocyte lineages at the expense of osteoblastogenesis, or by independent effects on the regulation of both osteoblastogenesis and adipogenesis.

Investigation of central versus peripheral effects of estradiol in ovariectomized mice.

It is generally believed that estrogens exert their bone sparing effects directly on the cells within the bone compartment. The aim of the present study was to investigate if central mechanisms might be involved in the bone sparing effect of estrogens. The dose-response of central (i.c.v) 17beta-estradiol (E2) administration was compared with that of peripheral (s.c.) administration in ovariectomized (ovx) mice. The dose-response curves for central and peripheral E2 administration did not differ for any of the studied estrogen-responsive tissues, indicating that these effects were mainly peripheral. In addition, ovx mice were treated with E2 and/or the peripheral estrogen receptor antagonist ICI 182,780. ICI 182,780 attenuated most of the estrogenic response regarding uterus weight, retroperitoneal fat weight, cortical BMC and trabecular bone mineral content (P<0.05). These findings support the notion that the primary target tissue that mediates the effect of E2 on bone is peripheral and not central.

Estrogen receptor (ER)-beta reduces ERalpha-regulated gene transcription, supporting a "ying yang" relationship between ERalpha and ERbeta in mice.

Estrogen is of importance for the regulation of adult bone metabolism. The aim of the present study was to determine the role of estrogen receptor-beta (ERbeta) in vivo on global estrogen-regulated transcriptional activity in bone. The effect of estrogen in bone of ovariectomized mice was determined using microarray analysis including 9400 genes. Most of the genes (95% = 240 genes) that were increased by estrogen in wild-type (WT) mice were also increased by estrogen in ERbeta-inactivated mice. Interestingly, the average stimulatory effect of estrogen on the mRNA levels of these genes was 85% higher in ERbeta-inactivated than in WT mice, demonstrating that ERbeta reduces estrogen receptor-alpha (ERalpha)-regulated gene transcription in bone. The average stimulatory effect of estrogen on estrogen-regulated bone genes in ERalpha-inactivated mice was intermediate between that seen in WT and ERalphabeta double-inactivated mice. Thus, ERbeta inhibits ERalpha-mediated gene transcription in the presence of ERalpha, whereas, in the absence of ERalpha, it can partially replace ERalpha. In conclusion, our in vivo data indicate that an important physiological role of ERbeta is to modulate ERalpha-mediated gene transcription supporting a "Ying Yang" relationship between ERalpha and ERbeta in 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 17beta-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 beta (TGF-beta), 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 alpha (ER-alpha)-mediated (5/7) while some genes (2/7) were regulated both via ER-alpha and ER-beta. 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.

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-alpha and ER-beta. The aim of this study was to investigate the relative roles of ER-alpha, ER-beta, and AR in the maintenance of trabecular bone in male mice. Seven-month-old male mice, lacking ER-alpha (ERKO), ER-beta (BERKO), or both receptors (DERKO), were orchidectomized (orx) and treated for 3 weeks with 0.7 microg/mouse per day of 17beta-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-a nor ER-beta 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-alpha. These results indicate that AR and ER-alpha are redundant in the maintenance of the trabecular bone in male mice. In contrast, ER-beta is of no importance for the regulation of trabecular bone in male mice.

Estrogen receptor alpha, but not estrogen receptor beta, is involved in the regulation of the hair follicle cycling as well as the thickness of epidermis in male mice.

Estrogen is of importance for the regulation of hair growth and epidermal thickness. The effects of estrogen have predominantly been studied in females; however, recent studies demonstrate that estrogen also is critical for males. The aim of this study was to investigate the relative functional importance of estrogen receptor alpha and estrogen receptor beta in the regulation of the hair follicle cycling and epidermal thickness in male mice. Seven month old transgenic male mice, lacking estrogen receptor alpha (ERKO), estrogen receptor beta (BERKO), or both receptors (DERKO), were orchidectomized and treated for 3 week with 17beta-estradiol or vehicle. Orchidectomy induced a synchronized anagen phase of the hair follicles, which was inhibited by 17beta-estradiol treatment in wild-type and BERKO mice, but not in ERKO and DERKO mice. Furthermore, 17beta-estradiol treatment increased the thickness of epidermis in wild-type and BERKO mice, but not in ERKO and DERKO. This study demonstrates that estrogen is of importance for the regulation of hair follicle cycling and epidermal thickness in male mice. The effect on hair follicle cycling is caused by an estrogen receptor alpha mediated inhibition of telogen-anagen transition and the effect of estrogen to increase epidermal thickness is associated with an estrogen receptor alpha mediated increase in the proliferative rate of the keratinocytes in the basal cell layer of the epidermis.

Estrogen increases coagulation factor V mRNA levels via both estrogen receptor-alpha and -beta in murine bone marrow/bone.

OBJECTIVES: Both oral estrogen-based hormone-replacement therapy and contraceptives increase the risk of venous thromboembolism. Several circulating factors involved in coagulation/fibrinolysis are expressed mainly in the liver whilst some are expressed in extrahepatic tissues, including bone marrow. The aim of this study was to identify estrogen-responsive target genes involved in the pathogenesis of estrogen-induced venous thromboembolism. METHODS: Ovariectomized mice were treated with 17beta-estradiol and possible effects on the expression of genes related to coagulation/fibrinolysis were investigated using DNA microarray analyses. RESULTS: None of the selected genes was regulated by 17beta-estradiol in the liver. Interestingly, 17beta-estradiol increased mRNA levels of coagulation factor V in the bone marrow/bone. Furthermore, this stimulatory effect of 17beta-estradiol on coagulation factor V expression can be mediated via both estrogen receptor-alpha and -beta. CONCLUSIONS: The expression of bone marrow-derived, but not liver-derived, coagulation factor V is increased by estrogen treatment in mice. The pathophysiological importance of this finding for estrogen-induced venous thromboembolism remains to be determined.

Dihydrotestosterone treatment results in obesity and altered lipid metabolism in orchidectomized mice.

OBJECTIVE: To determine the role of androgen receptor (AR) activation for adipose tissue metabolism. Sex steroids are important regulators of adipose tissue metabolism in men. Androgens may regulate the adipose tissue metabolism in men either directly by stimulation of the AR or indirectly by aromatization of androgens into estrogens and, thereafter, by stimulation of the estrogen receptors. Previous studies have shown that estrogen receptor alpha stimulation results in reduced fat mass in men. RESEARCH METHODS AND PROCEDURES: Orchidectomized mice were treated with the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT), 17beta-estradiol, or vehicle. Vo(2), Vco(2), resting metabolic rate, locomotor activity, and food consumption were measured. Furthermore, changes in hepatic gene expression were analyzed. RESULTS: DHT treatment resulted in obesity, associated with reduced energy expenditure and fat oxidation. In contrast, DHT did not affect food consumption or locomotor activity. Furthermore, DHT treatment resulted in increased high-density lipoprotein-cholesterol and triglyceride levels associated with markedly decreased 7alpha-hydroxylase gene expression, indicating decreased bile acid production. DISCUSSION: We showed that AR activation results in obesity and altered lipid metabolism in orchidectomized mice. One may speculate that AR antagonists might be useful in the treatment of obesity in men.