Membrane estrogen receptor α signaling modulates the sensitivity to estradiol treatment in a dose- and tissue- dependent manner.

Estradiol (E2) affects both reproductive and non-reproductive tissues, and the sensitivity to different doses of E2 varies between tissues. Membrane estrogen receptor α (mERα)-initiated signaling plays a tissue-specific role in mediating E2 effects, however, it is unclear if mERα signaling modulates E2 sensitivity. To determine this, we treated ovariectomized C451A females, lacking mERα signaling, and wildtype (WT) littermates with physiological (0.05 µg/mouse/day (low); 0.6 µg/mouse/day (medium)) or supraphysiological (6 µg/mouse/day (high)) doses of E2 (17ß-estradiol-3-benzoate) for three weeks. Low-dose treatment increased uterus weight in WT, but not C451A mice, while non-reproductive tissues (gonadal fat, thymus, trabecular and cortical bone) were unaffected in both genotypes. Medium-dose treatment increased uterus weight and bone mass and decreased thymus and gonadal fat weights in WT mice. Uterus weight was also increased in C451A mice, but the response was significantly attenuated (- 85%) compared to WT mice, and no effects were triggered in non-reproductive tissues. High-dose treatment effects in thymus and trabecular bone were significantly blunted (- 34% and - 64%, respectively) in C451A compared to WT mice, and responses in cortical bone and gonadal fat were similar between genotypes. Interestingly, the high dose effect in uterus was enhanced (+ 26%) in C451A compared to WT mice. In conclusion, loss of mERα signaling reduces the sensitivity to physiological E2 treatment in both non-reproductive tissues and uterus. Furthermore, the E2 effect after high-dose treatment in uterus is enhanced in the absence of mERα, suggesting a protective effect of mERα signaling in this tissue against supraphysiological E2 levels.

Glucocorticoids employ the monomeric glucocorticoid receptor to potentiate vitamin D3 and parathyroid hormone-induced osteoclastogenesis.

Glucocorticoid (GC) therapy decreases bone mass and increases the risk of fractures. We investigated interactions between the GC dexamethasone (DEX) and the bone resorptive agents 1,25(OH)2-vitamin D3 (D3) and parathyroid hormone (PTH) on osteoclastogenesis. We observed a synergistic potentiation of osteoclast progenitor cell differentiation and formation of osteoclasts when DEX was added to either D3- or PTH-treated mouse bone marrow cell (BMC) cultures. Cotreatment of DEX with D3 or PTH increased gene encoding calcitonin receptor (Calcr), acid phosphatase 5, tartrate resistant (Acp5), cathepsin K (Ctsk), and TNF superfamily member 11 (Tnfsf11) mRNA, receptor activator of NF-κB ligand protein (RANKL), numbers of osteoclasts on plastic, and pit formation and release of C-terminal fragment of type I collagen from cells cultured on bone slices. Enhanced RANKL protein expression caused by D3 and DEX was absent in BMC from mice in which the GC receptor (GR) was deleted in stromal cells/osteoblasts. Synergistic interactions between DEX and D3 on RANKL and osteoclast formation were present in BMC from mice with attenuated GR dimerization. These data demonstrate that the GR cooperates with D3 and PTH signaling, causing massive osteoclastogenesis, which may explain the rapid bone loss observed with high dosages of GC treatment.-Conaway, H. H., Henning, P., Lie, A., Tuckermann, J., Lerner, U. H. Glucocorticoids employ the monomeric glucocorticoid receptor to potentiate vitamin D3 and parathyroid hormone-induced osteoclastogenesis.

Clinically relevant doses of vitamin A decrease cortical bone mass in mice

Excess vitamin A has been associated with decreased cortical bone thickness and increased fracture risk. While most studies in rodents have employed high dosages of vitamin A for short periods of time, we investigated the bone phenotype in mice after longer exposure to more clinically relevant doses. For 1, 4 and 10 weeks, mice were fed a control diet (4.5 µg retinyl acetate/g chow), a diet modeled from the human upper tolerable limit (UTL; 20 µg retinyl acetate/g chow) and a diet three times UTL (supplemented; 60 µg retinyl acetate/g chow). Time-dependent decreases in periosteal circumference and bone mineral content were noted with the supplemented dose. These reductions in cortical bone resulted in a significant time-dependent decrease of predicted strength and a non-significant trend toward reduced bone strength as analyzed by three-point bending. Trabecular bone in tibiae and vertebrae remained unaffected when vitamin A was increased in the diet. Dynamic histomorphometry demonstrated that bone formation was substantially decreased after 1 week of treatment at the periosteal site with the supplemental dose. Increasing amount of vitamin A decreased endocortical circumference, resulting in decreased marrow area, a response associated with enhanced endocortical bone formation. In the presence of bisphosphonate, vitamin A had no effect on cortical bone, suggesting that osteoclasts are important, even if effects on bone resorption were not detected by osteoclast counting, genes in cortical bone or analysis of serum TRAP5b and CTX. In conclusion, our results indicate that even clinically relevant doses of vitamin A have a negative impact on the amount of cortical bone.

Porcupine inhibitors impair trabecular and cortical bone mass and strength in mice.

WNT signaling is involved in the tumorigenesis of various cancers and regulates bone homeostasis. Palmitoleoylation of WNTs by Porcupine is required for WNT activity. Porcupine inhibitors are under development for cancer therapy. As the possible side effects of Porcupine inhibitors on bone health are unknown, we determined their effects on bone mass and strength. Twelve-week-old C57BL/6N female mice were treated by the Porcupine inhibitors LGK974 (low dose = 3 mg/kg/day; high dose = 6 mg/kg/day) or Wnt-C59 (10 mg/kg/day) or vehicle for 3 weeks. Bone parameters were assessed by serum biomarkers, dual-energy X-ray absorptiometry, µCT and histomorphometry. Bone strength was measured by the 3-point bending test. The Porcupine inhibitors were well tolerated demonstrated by normal body weight. Both doses of LGK974 and Wnt-C59 reduced total body bone mineral density compared with vehicle treatment (P < 0.001). Cortical thickness of the femur shaft (P < 0.001) and trabecular bone volume fraction in the vertebral body (P < 0.001) were reduced by treatment with LGK974 or Wnt-C59. Porcupine inhibition reduced bone strength in the tibia (P < 0.05). The cortical bone loss was the result of impaired periosteal bone formation and increased endocortical bone resorption and the trabecular bone loss was caused by reduced trabecular bone formation and increased bone resorption. Porcupine inhibitors exert deleterious effects on bone mass and strength caused by a combination of reduced bone formation and increased bone resorption. We suggest that cancer targeted therapies using Porcupine inhibitors may increase the risk of fractures.