VITAMIN A ENHANCED PERIOSTEAL OSTEOCLASTOGENESIS IS ASSOCIATED WITH INCREASED NUMBER OF TISSUE-DERIVED MACROPHAGES/OSTEOCLAST PROGENITORS.

A deleterious effect of elevated levels of vitamin A on bone health has been reported in numerous clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. These observations indicate that osteoclastogenesis on the endocortical and periosteal surfaces of bone is differentially controlled by vitamin A. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine RANKL, on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (EC50 ∼3nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as M-CSF receptor, RANK, F4/80 and CD11b, as well as by FACS-analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased M-CSF or IL-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes also in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

Toll-like receptor-2 induced inflammation causes local bone formation and activates canonical Wnt signaling.

It is well established that inflammatory processes in the vicinity of bone often induce osteoclast formation and bone resorption. Effects of inflammatory processes on bone formation are less studied. Therefore, we investigated the effect of locally induced inflammation on bone formation. Toll-like receptor (TLR) 2 agonists LPS from Porphyromonas gingivalis and PAM2 were injected once subcutaneously above mouse calvarial bones. After five days, both agonists induced bone formation mainly at endocranial surfaces. The injection resulted in progressively increased calvarial thickness during 21 days. Excessive new bone formation was mainly observed separated from bone resorption cavities. Anti-RANKL did not affect the increase of bone formation. Inflammation caused increased bone formation rate due to increased mineralizing surfaces as assessed by dynamic histomorphometry. In areas close to new bone formation, an abundance of proliferating cells was observed as well as cells robustly stained for Runx2 and alkaline phosphatase. PAM2 increased the mRNA expression of Lrp5, Lrp6 and Wnt7b, and decreased the expression of Sost and Dkk1. In situ hybridization demonstrated decreased Sost mRNA expression in osteocytes present in old bone. An abundance of cells expressed Wnt7b in Runx2-positive osteoblasts and ß-catenin in areas with new bone formation. These data demonstrate that inflammation, not only induces osteoclastogenesis, but also locally activates canonical WNT signaling and stimulates new bone formation independent on bone resorption.

Phosphorylation of S122 in ERα is important for the skeletal response to estrogen treatment in male mice.

Estrogen receptor alpha (ERα) signaling has beneficial skeletal effects in males. ERα signaling also affects other tissues, and to find bone-specific treatments, more knowledge regarding tissue-specific ERα signaling is needed. ERα is subjected to posttranslational modifications, including phosphorylation, which can influence ERα function in a tissue-specific manner. To determine the importance of phosphorylation site S122 (corresponding to human ERα site S118) for the skeleton and other tissues, male mice with a S122A mutation were used. Total areal bone mineral density was similar between gonadal intact S122A and WT littermates followed up to 12 months of age, and weights of estrogen-responsive organs normalized for body weight were unchanged between S122A and WT males at both 3 and 12 months of age. Interestingly, 12-month-old S122A males had decreased body weight compared to WT. To investigate if site S122 affects the estrogen response in bone and other tissues, 12-week-old S122A and WT males were orchidectomized (orx) and treated with estradiol (E2) or placebo pellets for four weeks. E2 increased cortical thickness in tibia in both orx WT (+ 60%, p < 0.001) and S122A (+ 45%, p < 0.001) males. However, the E2 effect on cortical thickness was significantly decreased in orx S122A compared to WT mice (- 24%, p < 0.05). In contrast, E2 affected trabecular bone and organ weights similarly in orx S122A and WT males. Thus, ERα phosphorylation site S122 is required for a normal E2 response specifically in cortical bone in male mice, a finding that may have implications for development of future treatments against male osteoporosis.

Arginine site 264 in murine estrogen receptor-α is dispensable for the regulation of the skeleton.

Mutation of arginine 264 in ERα has been shown to abrogate rapid membrane ERα-mediated endothelial effects. Our novel finding that mutation of R264 is dispensable for ERα-mediated skeletal effects supports the concept that R264 determines tissue specificity of ERα. Estrogen protects against bone loss but is not a suitable treatment due to adverse effects in other tissues. Therefore, increased knowledge regarding estrogen signaling in estrogen-responsive tissues is warranted to aid the development of bone-specific estrogen treatments. Estrogen receptor-α (ERα), the main mediator of estrogenic effects in bone, is widely subjected to posttranslational modifications (PTMs). In vitro studies have shown that methylation at site R260 in the human ERα affects receptor localization and intracellular signaling. The corresponding amino acid R264 in murine ERα has been shown to have a functional role in endothelium in vivo, although the methylation of R264 in the murine gene is yet to be empirically demonstrated. The aim of this study was to investigate whether R264 in ERα is involved in the regulation of the skeleton in vivo. Dual-energy X-ray absorptiometry (DEXA) analysis at 3, 6, 9, and 12 mo of age showed no differences in total body areal bone mineral density (BMD) between R264A and wild type (WT) in either female or male mice. Furthermore, analyses using computed tomography (CT) demonstrated that trabecular bone mass in tibia and vertebra and cortical thickness in tibia were similar between R264A and WT mice. In addition, R264A females displayed a normal estrogen treatment response in trabecular bone mass as well as in cortical thickness. Furthermore, uterus, thymus, and adipose tissue responded similarly in R264A and WT female mice after estrogen treatment. In conclusion, our novel finding that mutation of R264 in ERα does not affect the regulation of the skeleton, together with the known role of R264 for ERα-mediated endothelial effects, supports the concept that R264 determines tissue specificity of ERα.NEW & NOTEWORTHY Mutation of arginine 264 in ERα has been shown to abrogate rapid membrane ERα-mediated endothelial effects. Our novel finding that mutation of R264 is dispensable for ERα-mediated skeletal effects supports the concept that R264 determines tissue specificity of ERα.

Phosphorylation site S122 in estrogen receptor α has a tissue-dependent role in female mice.

Estrogen treatment increases bone mass and reduces fat mass but is associated with adverse effects in postmenopausal women. Knowledge regarding tissue-specific estrogen signaling is important to aid the development of new tissue-specific treatments. We hypothesized that the posttranslational modification phosphorylation in estrogen receptor alpha (ERα) may modulate ERα activity in a tissue-dependent manner. Phosphorylation of site S122 in ERα has been shown in vitro to affect ERα activity, but the tissue-specific role in vivo is unknown. We herein developed and phenotyped a novel mouse model with a point mutation at the phosphorylation site 122 in ERα (S122A). Female S122A mice had increased fat mass and serum insulin levels but unchanged serum sex steroid levels, uterus weight, bone mass, thymus weight, and lymphocyte maturation compared to WT mice. In conclusion, phosphorylation site S122 in ERα has a tissue-dependent role with an impact specifically on fat mass in female mice. This study is the first to demonstrate in vivo that a phosphorylation site in a transactivation domain in a nuclear steroid receptor modulates the receptor activity in a tissue-dependent manner.

Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.

Increased vitamin A consumption is associated with decreased cortical bone mass and increased fracture risk in humans. Rodent studies have demonstrated that hypervitaminosis A increases cortical bone resorption, whereas the importance of the effects on bone formation is less well defined. We used an experimental model of increased bone formation by loading of the tibiae to investigate the effect of vitamin A on bone formation. Control [retinol activity equivalents (RAE) 4.5 µg/g chow] or vitamin A (RAE 60 µg/g chow) diets were given to female C57BL/6N mice for 4 wk, after which the tibiae were subjected to axial loading on alternate days for 2 wk, while the diets were continued. Vitamin A inhibited the loading-induced increase in trabecular and cortical bone volume. This was attributed to inhibition of loading-induced increase in osteoblast number and activity, and expression of osteoblastic genes Sp7, Alpl, and Col1a1 in cortical bone. Vitamin A, loading, and combination thereof also resulted in site-specific effects on bone composition measured by Raman spectroscopy. In summary, a clinically relevant dose of vitamin A suppresses the loading-induced gain of bone mass by decreasing bone formation. These observations may have implications for regulation of bone mass caused by physical activity and the risk of osteoporosis in humans.-Lionikaite, V., Henning, P., Drevinge, C., Shah, F. A., Palmquist, A., Wikström, P., Windahl, S. H., Lerner, U. H. Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.

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.

Effects of retinoids on physiologic and inflammatory osteoclastogenesis in vitro.

Increased intake of vitamin A (retinoids) is associated with decreased bone mass and increased fracture risk in humans. Mechanistic studies in rodents have shown that hypervitaminosis A results in decreased bone mass caused by an increase in periosteal osteoclasts while simultaneously decreasing endocortic osteoclasts. In vivo and ex vivo bone organ cultures have demonstrated that excess retinoids increase osteoclast formation due to increased receptor activator of nuclear factor kappa B-ligand (RANKL) expression. In vitro, studies using murine bone marrow macrophages (BMM) have shown that retinoids inhibit osteoclast formation induced by recombinant RANKL. These opposing in vivo/ex vivo versus in vitro effects may elucidate why excess retinoids affect periosteal and endocortic osteoclast formation differently. In addition, it has been reported that retinoids can inhibit osteoclast formation under inflammatory conditions such as experimentally induced arthritis in mice. In the present study, we have compared the effect of all-trans-retinoic acid (ATRA) on physiologically and inflammatory induced osteoclastogenesis. ATRA inhibited physiologically induced (RANKL) osteoclast formation of human peripheral blood monocytes and mouse BMM as well as human monocytes stimulated with the pro-inflammatory compounds, TNF-α and LPS. The inhibition was due to impeded differentiation, rather than fusion, of mononucleated progenitor cells. ATRA disrupted differentiation by interfering with osteoclastogenic intracellular signaling. In line with this view, overexpression of Tnfrsf11a (encodes for RANK) in BMM could not overcome the inhibition of osteoclastogenesis by ATRA. The data suggest that ATRA inhibits both physiologic and inflammatory osteoclast differentiation of progenitors from the bone marrow and peripheral blood.

Genome-wide association of multiple complex traits in outbred mice by ultra-low-coverage sequencing.

Two bottlenecks impeding the genetic analysis of complex traits in rodents are access to mapping populations able to deliver gene-level mapping resolution and the need for population-specific genotyping arrays and haplotype reference panels. Here we combine low-coverage (0.15×) sequencing with a new method to impute the ancestral haplotype space in 1,887 commercially available outbred mice. We mapped 156 unique quantitative trait loci for 92 phenotypes at a 5% false discovery rate. Gene-level mapping resolution was achieved at about one-fifth of the loci, implicating Unc13c and Pgc1a at loci for the quality of sleep, Adarb2 for home cage activity, Rtkn2 for intensity of reaction to startle, Bmp2 for wound healing, Il15 and Id2 for several T cell measures and Prkca for bone mineral content. These findings have implications for diverse areas of mammalian biology and demonstrate how genome-wide association studies can be extended via low-coverage sequencing to species with highly recombinant outbred populations.