Cystatin M/E ameliorates bone resorption through increasing osteoclastic cell estrogen influx.

In multiple myeloma (MM), increased osteoclast differentiation leads to the formation of osteolytic lesions in most MM patients. Bisphosphonates, such as zoledronic acid (ZA), are used to ameliorate bone resorption, but due to risk of serious side effects as well as the lack of repair of existing lesions, novel anti-bone resorption agents are required. Previously, the absence of osteolytic lesions in MM was strongly associated with elevated levels of cystatin M/E (CST6), a cysteine protease inhibitor, secreted by MM cells. In this study, both MM- and ovariectomy (OVX)-induced osteoporotic mouse models were used to compare the effects of recombinant mouse CST6 (rmCst6) and ZA on preventing bone loss. µCT showed that rmCst6 and ZA had similar effects on improving percent bone volume, and inhibited differentiation of non-adherent bone marrow cells into mature osteoclasts. Single-cell RNA sequencing showed that rmCst6 and not ZA treatment reduced bone marrow macrophage percentage in the MM mouse model compared to controls. Protein and mRNA arrays showed that both rmCst6 and ZA significantly inhibit OVX-induced expression of inflammatory cytokines. For OVX mice, ERα protein expression in bone was brought to sham surgery level by only rmCst6 treatments. rmCst6 significantly increased mRNA and protein levels of ERα and significantly increased total intracellular estrogen concentrations for ex vivo osteoclast precursor cell cultures. Based on these results, we conclude that CST6 improves MM or OVX bone loss models by increasing the expression of estrogen receptors as well as the intracellular estrogen concentration in osteoclast precursors, inhibiting their maturation.

Phenolic acids prevent sex-steroid deficiency-induced bone loss and bone marrow adipogenesis in mice.

Phenolic acids, such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA), can be produced from microbiome digestion of polyphenols. Previously it was found that HA and 3-3-PPA facilitate bone formation and suppress bone resorption. However, the mechanism of action by which HA and 3-3-PPA protect bone from degeneration is currently unknown. In this report, we present that HA and 3-3-PPA suppression of bone resorption is able to ameliorate bone loss in an ovariectomy (OVX) osteopenic mouse model though not to the extent of Zoledronic acid (ZA). HA and 3-3-PPA treatments were shown to significantly decrease bone marrow adipocyte-like cell formation and inhibited gene expression of key adipogenesis regulator peroxisome proliferator activated receptor gamma (PPARγ) and lipoprotein lipase (Lpl) in bone from OVX mice. In addition, ChIP experiments showed that the association between PPARγ and Lpl promoter region in preadipocyte-like cells was significantly suppressed following HA or 3-3-PPA treatment. Contrasting HA and 3-3-PPA, ZA significantly increased TRAP activity in the area close to growth plate and significantly suppressed bone cell proliferation. These data suggest that phenolics acids such as HA or 3-3-PPA may prevent bone degeneration after OVX through suppression of inflammatory milieu in the bone.

Decreased bone resorption in Ezh2 myeloid cell conditional knockout mouse model.

Osteoclasts derived from hematopoietic stem cells control bone resorption. Identifying novel molecules that can epigenetically regulate osteoclastogenesis is important for developing novel treatments for osteoporosis and other disorders associated with bone deterioration and promoting healthy bone formation. The polycomb group (PcG) protein enhancer of zeste homolog 2 (Ezh2), a histone lysine methyltransferase, is associated with epigenetic regulation of numerous cellular processes, but its involvement in bone cell development and homeostasis is not yet clear. Here, LysM-Cre mice were crossed with Ezh2flox/flox mice to delete Ezh2 in myeloid cell lineage mature macrophages. Conditional knockout of Ezh2 (CKO) in myeloid cell line resulted in significant increases in postnatal bone growth in the first 6 months of life for both male and female mice. For female mice, optimal bone mass was seen for mice with Ezh2 deleted in both chromosomes in a pair (f/f Cre+ ; CKO). For male mice, optimal bone mass was found after deletion of Ezh2 from just one chromosome (f/- Cre+ ) with no difference in bone phenotype between f/- Cre+ and CKO male mice. In addition to the gender-specific difference in bone phenotype, Ezh2 CKO mice had significantly less macrophages (CD11b+) present in the bone marrow compared with control mice as well as significantly more mature osteoblasts and bone formation biomarkers present (P1NP, osteocalcin). Inflammatory array for protein lysed from bone tissue revealed deletion of Ezh2 decreased inflammatory milieu in both male and female mice compared with controls. Unexpectedly, myeloid cell deletion of Ezh2 also increased the number of mature osteoblast present in the bone. Deletion of Ezh2 also led to an increase in gene expression of osteoclast-suppressive genes IRF8, MafB, and Arg1 due to a decrease in the presence of the suppressive H3K27me3 epigenetic mark. These findings suggest that manipulation of Ezh2 expression may be a viable strategy to combat bone resorptive disorders such as osteoporosis or arthritis.

Short-Term Increased Physical Activity During Early Life Affects High-Fat Diet-Induced Bone Loss in Young Adult Mice.

Mechanical stresses associated with physical activity (PA) have beneficial effects on increasing BMD and improving bone quality. However, a high-fat diet (HFD) and obesity tend to have negative effects on bone, by increasing bone marrow adiposity leading to increased excretion of proinflammatory cytokines, which activate RANKL-induced bone resorption. In the current study, whether short-term increased PA via access to voluntary wheel running during early life has persistent and protective effects on HFD-induced bone resorption was investigated. Sixty 4-week-old male C57BL6/J mice were divided into two groups postweaning: without or with PA (access to voluntary running wheel 7-8 km/day) for 4 weeks. After 4 weeks with or without PA, mice were further subdivided into control diet or HFD groups for 8 weeks, and then all animals were switched back to control diet for an additional 4 weeks. Mice from the HFD groups were significantly heavier and obese; however, after 4 weeks of additional control diet their body weights returned to levels of mice on continuous control diet. Using µ-CT and confirmed by pQCT of tibias and spines ex vivo, it was determined that bone volume and trabecular BMD were significantly increased with PA in control diet animals compared with sedentary animals without access to wheels, and such anabolic effects of PA on bone were sustained after ceasing PA in adult mice. Eight weeks of a HFD deteriorated bone development in mice. Unexpectedly, early-life PA did not prevent persistent effects of HFD on deteriorating bone quality; in fact, it exacerbated a HFD-induced inflammation, osteoclastogenesis, and trabecular bone loss in adult mice. In accordance with these data, signal transduction studies revealed that a HFD-induced Ezh2, DNA methyltransferase 3a, and nuclear factor of activated T-cells 1 expression were amplified in nonadherent hematopoietic cells. In conclusion, short-term increased PA in early life is capable of increasing bone mass; however, it alters the HFD-induced bone marrow hematopoietic cell-differentiation program to exacerbate increased bone resorption if PA is halted. © 2021 Arkansas Children's Nutrition Center. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Hippuric acid and 3-(3-hydroxyphenyl) propionic acid inhibit murine osteoclastogenesis through RANKL-RANK independent pathway.

Nutritional factors influence bone development. Previous studies demonstrated that bone mass significantly increased with suppressed bone resorption in early life of rats fed with AIN-93G semi-purified diets supplemented with 10% whole blueberry (BB) powder for 2 weeks. However, the effects of increased phenolic acids in animal serum due to this diet on bone and bone resorption were unclear. This in vitro and in ex vivo study examined the effects of phenolic hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on osteoclastic cell differentiation and bone resorption. We cultured murine osteoclast (macrophage) cell line, RAW 264.7 cells, and hematopoietic osteoclast progenitor cells (isolated from 4-week-old C57BL6/J mice) with 50 ng/ml of receptor activator of nuclear factor κ-Β ligand (RANKL). Morphologic studies showed decreased osteoclast number with treatment of 2.5% mouse serum from BB diet-fed animals compared with those treated with serum from standard casein diet-fed mice in both RAW 264.7 cell and primary cell cultures. HA and 3-3-PPA, but not 3-4-PPA, had dose-dependent suppressive effects on osteoclastogenesis and osteoclast resorptive activity in Corning osteo-assay plates. Signaling pathway analysis showed that after pretreatment with HA or 3-3-PPA, RANKL-stimulated increase of osteoclastogenic markers, such as nuclear factor of activated T-cells, cytoplasmic 1 and matrix metallopeptidase 9 gene/protein expression were blunted. Inhibitory effects of HA and 3-3-PPA on osteoclastogenesis utilized RANKL/RANK independent mediators. The study revealed that HA and 3-3-PPA significantly inhibited osteoclastogenesis and bone osteoclastic resorptive activity.

3-(3-Hydroxyphenyl)-Propionic Acid (PPA) Suppresses Osteoblastic Cell Senescence to Promote Bone Accretion in Mice.

Phenolic acids (PAs) are metabolites derived from polyphenolic compounds found in fruits and vegetables resulting from the actions of gut bacteria. Previously, we reported that the levels of seven individual PAs were found to be at least 10 times higher in the serum of rats fed a blueberry (BB)-containing diet compared to those fed a control diet. We have characterized the effects of one such BB-associated serum PA, 3-(3-hydroxyphenyl)-propionic acid (PPA), on senescence signaling and promotion of mesenchymal stem cell differentiation toward osteoblasts, while suppressing adipogenesis in the stem cells. To better understand the mechanistic actions of PPA on bone formation in vivo, we administered four doses of PPA (0.1, 0.5, 1, and 5 mg/kg/day; daily i.p.) to 1-month-old female C57BL6/J mice for 30 days. We did not observe significant effects of PPA on cortical bone; however, there were significantly higher bone volume and trabecular thickness and increased osteoblastic cell number, but decreased osteoclastic cell number in PPA-treated groups compared to controls. These morphological and cellular outcomes of bone were reflected in changes of bone formation markers in serum and bone marrow plasma. PPA treatment reduced senescence signaling as evaluated by senescence-associated ß-galactosidase activity, PPARγ, p53, and p21 expression in bone. In conclusion, PPA is capable of altering the mesenchymal stem cell differentiation program and bone cell senescence. This raises the possibility that BB-rich diets promote bone growth through increasing systemic PAs, a question that merits additional investigation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Dietary factors during early life program bone formation in female rats.

Nutritional status during intrauterine and early postnatal life impacts the risk of chronic diseases; however, evidence for an association between early-life dietary factors and bone health in adults is limited. Soy protein isolate (SPI) may be one such dietary factor that promotes bone accretion during early life with persistent effects into adulthood. In the present study, we fed postnatal day (PND) 24 weanling female rats an SPI diet for 30 d [short-term SPI (ST-SPI)], and on PND 55, we switched SPI diet to control Cas diet until age 6 mo. Rats then underwent either ovariectomy (OVX) or sham surgery and thereafter either continued to be fed an SPI diet or control diet for 1 or 3 wk. We showed significantly increased bone mass in 30-d SPI-fed young rats compared with controls. OVX-induced bone loss was associated with increased osteoblastic cell senescence. On the one hand, both long-term SPI (continuous SPI diet throughout life) and ST-SPI diet only in early life protected against 1 wk post-OVX-associated bone loss. On the other hand, long-term SPI diet diminished the loss of total, trabecular, and cortical bone mineral density, whereas ST-SPI diet only reduced cortical bone mineral density loss 3 wk post-OVX. Persistent and protective effects of SPI diets on OVX-induced bone loss were associated with down-regulation of the caveolin-1/p53-mediated senescence pathway in bone. We recapitulated these results in cell cultures. Reprogramming of cellular senescence signaling by SPI-associated isoflavones in osteoblastic cells may explain the persistent effects of SPI on bone. These results suggest that OVX-induced bone loss, in part, is a result of increased osteoblastic cell senescence, and that ST-SPI diet early in life has modest but persistent programming effects on bone formation to prevent OVX-induced bone loss in adult female rats.-Chen, J.-R., Lazarenko, O. P., Blackburn, M. L., Shankar, K. Dietary factors during early life program bone formation in female rats.

Maternal Obesity Programs Senescence Signaling and Glucose Metabolism in Osteo-Progenitors From Rat and Human.

Nutritional status during intrauterine and early postnatal life impacts the risk of chronic diseases, presumably via epigenetic mechanisms. However, evidence on the impact of gestational events on regulation of embryonic bone cell fate is sparse. We investigated the effects of maternal obesity on fetal osteoblast development in both rodents and humans. Female rats were fed control or an obesogenic high-fat diet (HFD) for 12 weeks and mated with male rats fed control diets, and respective maternal diets were continued during pregnancy. Embryonic rat osteogenic calvarial cells (EOCCs) were taken from gestational day 18.5 fetuses from control and HFD dams. EOCCs from HFD obese dams showed increases in p53/p21-mediated cell senescence signaling but decreased glucose metabolism. Decreased aerobic glycolysis in HFD-EOCCs was associated with decreased osteoblastic cell differentiation and proliferation. Umbilical cord human mesenchymal stem cells (MSCs) from 24 pregnant women (12 obese and 12 lean) along with placentas were collected upon delivery. The umbilical cord MSCs of obese mothers displayed less potential toward osteoblastogenesis and more towards adipogenesis. Human MSCs and placenta from obese mothers also exhibited increased cell senescence signaling, whereas MSCs showed decreased glucose metabolism and insulin resistance. Finally, we showed that overexpression of p53 linked increased cell senescence signaling and decreased glucose metabolism in fetal osteo-progenitors from obese rats and humans. These findings suggest programming of fetal preosteoblastic cell senescence signaling and glucose metabolism by maternal obesity.

p47phox-Nox2-dependent ROS Signaling Inhibits Early Bone Development in Mice but Protects against Skeletal Aging.

Bone remodeling is age-dependently regulated and changes dramatically during the course of development. Progressive accumulation of reactive oxygen species (ROS) has been suspected to be the leading cause of many inflammatory and degenerative diseases, as well as an important factor underlying many effects of aging. In contrast, how reduced ROS signaling regulates inflammation and remodeling in bone remains unknown. Here, we utilized a p47(phox) knock-out mouse model, in which an essential cytosolic co-activator of Nox2 is lost, to characterize bone metabolism at 6 weeks and 2 years of age. Compared with their age-matched wild type controls, loss of Nox2 function in p47(phox-/-) mice resulted in age-related switch of bone mass and strength. Differences in bone mass were associated with increased bone formation in 6-week-old p47(phox-/-) mice but decreased in 2-year-old p47(phox-/-) mice. Despite decreases in ROS generation in bone marrow cells and p47(phox)-Nox2 signaling in osteoblastic cells, 2-year-old p47(phox-/-) mice showed increased senescence-associated secretory phenotype in bone compared with their wild type controls. These in vivo findings were mechanistically recapitulated in ex vivo cell culture of primary fetal calvarial cells from p47(phox-/-) mice. These cells showed accelerated cell senescence pathway accompanied by increased inflammation. These data indicate that the observed age-related switch of bone mass in p47(phox)-deficient mice occurs through an increased inflammatory milieu in bone and that p47(phox)-Nox2-dependent physiological ROS signaling suppresses inflammation in aging.

Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways.

It has been suggested that the beneficial effects of soy protein isolate (SPI) on bone quality are due to either stimulation of estrogenic signaling via isoflavones or through a novel and as yet uncharacterized nonestrogenic pathway. In our study, SPI-fed rat serum inhibited the osteoblastic cell senescence pathway. This effect was accompanied by stimulation of cell differentiation, proliferation, and significant restoration of replicative senescent bone marrow mesenchymal ST2 cells (passaged 30 times). These effects were reproduced in bone from 5-wk-old intact and 10-wk-old ovariectomized female rats fed SPI diets. Caveolin-1 and p53 expression was decreased in bone in SPI-fed, but not in 17ß-estradiol (E2)-treated rats. In cell culture studies, membranous caveolin-1 and nuclear p53 expression was greater in replicative senescent ST2 cell cultures than in earlier passaged cells. SPI-fed rat serum significantly down-regulated both caveolin-1 and p53 in senescent and nonsenescent cells. Replicative senescent ST2 cells exhibited a strong association among caveolin-1, p53, and mouse double minute 2 homologue (mdm2), which was inhibited by SPI-fed rat serum. Overexpression of caveolin-1 in ST2 cells resulted in increased expression of p53 and p21, whereas, knockdown of caveolin-1 using shRNA led to increases in mdm2 and eliminated SPI-fed rat serum's effects on p53 and p21 expression. In contrast, manipulation of caveolin-1 expression did not affect the actions of E2 or isoflavones on p53 expression in either ST2 or OB6 cells. These results suggest that caveolin-1 is a mediator of nonestrogenic SPI effects on bone cells.-Zhang, J., Lazarenko, O. P., Blackburn, M. L., Badger, T. M., Ronis, M. J. J., Chen, J.-R. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways.

Diet-derived phenolic acids regulate osteoblast and adipocyte lineage commitment and differentiation in young mice.

A blueberry (BB)-supplemented diet has been previously shown to significantly stimulate bone formation in rapidly growing male and female rodents. Phenolic acids (PAs) are metabolites derived from polyphenols found in fruits and vegetables as a result of the actions of gut bacteria, and they were found in the serum of rats fed BB-containing diet. We conducted in vitro studies with PAs and demonstrated stimulation of osteoblast differentiation and proliferation. On the other hand, adipogenesis was inhibited. To more fully understand the mechanistic actions of PAs on bone formation, we administered hippuric acid, one of the major metabolites found in animal circulation after BB consumption, to prepubertal female mice for 2 weeks. We found that hippuric acid was able to stimulate bone-forming gene expression but suppress PPARγ expression, leading to increased bone mass dose-dependently. Cellular signaling studies further suggested that the skeletal effects of PAs appeared to be mediated through activation of G-protein-coupled receptor 109A and downstream p38 MAP kinase and osterix. In conclusion, PAs are capable of altering the mesenchymal stem cell differentiation program and merit investigation as potential dietary therapeutic alternatives to drugs for degenerative bone disorders. © 2014 American Society for Bone and Mineral Research.

Feeding blueberry diets to young rats dose-dependently inhibits bone resorption through suppression of RANKL in stromal cells.

Previous studies have demonstrated that weanling rats fed AIN-93G semi-purified diets supplemented with 10% whole blueberry (BB) powder for two weeks beginning on postnatal day 21 (PND21) significantly increased bone formation at PND35. However, the minimal level of dietary BB needed to produce these effects is, as yet, unknown. The current study examined the effects of three different levels of BB diet supplementation (1, 3, and 5%) for 35 days beginning on PND25 on bone quality, and osteoclastic bone resorption in female rats. Peripheral quantitative CT scan (pQCT) of tibia, demonstrated that bone mineral density (BMD) and content (BMC) were dose-dependently increased in BB-fed rats compared to controls (P<0.05). Significantly increased bone mass after feeding 5% BB extracts was also observed in a TEN (total enteral nutrition) rat model in which daily caloric and food intake was precisely controlled. Expression of RANKL (receptor activator of nuclear factor-κB ligand) a protein essential for osteoclast formation was dose-dependently decreased in the femur of BB animals. In addition, expression of PPARγ (peroxisome proliferator-activated receptor γ) which regulates bone marrow adipogenesis was suppressed in BB diet rats compared to non-BB diet controls. Finally, a set of in vitro cell cultures revealed that the inhibitory effect of BB diet rat serum on RANKL expression was more profound in mesenchymal stromal cells compared to its effect on mature osteoblasts, pre-adipocytes and osteocytes. These results suggest that inhibition of bone resorption may contribute to increased bone mass during early development after BB consumption.

Vitamin D supplementation protects against bone loss associated with chronic alcohol administration in female mice.

Chronic alcohol abuse results in decreased bone mineral density (BMD), which can lead to increased fracture risk. In contrast, low levels of alcohol have been associated with increased BMD in epidemiological studies. Alcohol's toxic skeletal effects have been suggested to involve impaired vitamin D/calcium homeostasis. Therefore, dietary vitamin D supplementation may be beneficial in reducing bone loss associated with chronic alcohol consumption. Six-week-old female C57BL/6J mice were pair-fed ethanol (EtOH)-containing liquid diets (10 or 36% total calories) for 78 days. EtOH exposure at 10% calories had no effects on any measured bone or serum parameter. EtOH consumption at 36% of calories reduced BMD and bone strength (P<0.05), decreased osteoblastogenesis, increased osteoclastogenesis, suppressed 1,25-hydroxyvitamin D3 [1,25(OH)2D3] serum concentrations (P<0.05), and increased apoptosis in bone cells compared with pair-fed controls. In a second study, female mice were pair-fed 30% EtOH diets with or without dietary supplementation with vitamin D3 (cholecalciferol; VitD) for 40 days. VitD supplementation in the EtOH diet protected against cortical bone loss, normalized alcohol-induced hypocalcaemia, and suppressed EtOH-induced expression of receptor of nuclear factor-κB ligand mRNA in bone. In vitro, pretreatment of 1,25(OH)2D3 in osteoblastic cells inhibited EtOH-induced apoptosis. In EtOH/VitD mice circulating 1,25(OH)2D3 was lower compared with mice receiving EtOH alone (P<0.05), suggesting increased sensitivity to feedback control of VitD metabolism in the kidney. These findings suggest dietary VitD supplementation may prevent skeletal toxicity in chronic drinkers by normalizing calcium homeostasis, preventing apoptosis, and suppressing EtOH-induced increases in bone resorption.

Differential effects of short term feeding of a soy protein isolate diet and estrogen treatment on bone in the pre-pubertal rat.

BACKGROUND: Previous reports suggest that beneficial effects of soy on bone quality are due to the estrogenic actions of isoflavone phytochemicals associated with the protein. However, mechanistic studies comparing the effects of soy diet and estrogens on bone, particularly in rapidly growing animals are lacking. METHODOLOGY AND PRINCIPAL FINDINGS: We studied the effects of short term feeding of soy protein isolate (SPI) on bone in comparison to the effects of 17ß-estradiol (E2) in pre-pubertal rats. Female rats were weaned to one of 4 treatments: 1) a control casein-based diet (CAS); 2) CAS with subcutaneous E2 (10 µg/kg/d) (CAS+E2); 3) a SPI-containing diet (SPI); or 4) SPI with subcutaneous E2 (SPI) or SPI with 10 µg/kg/d E2 (SPI+E2) for 14 days beginning on postnatal day 20. SPI increased while E2 decreased bone turnover compared to CAS. In contrast, both treatments decreased serum sclerostin levels. Microarray analysis of RNA isolated from bone revealed 652 genes regulated by SPI, 491 genes regulated by E2, and 266 genes regulated by both SPI diet and E2 compared to CAS. The expression of caveolin-1, a protein localized in the cell membrane, was down-regulated (p<0.05) in rats fed SPI, but not by E2 compared to rats fed casein. Down-regulation of caveolin-1 by SPI was associated with increased BMP2, Smad and Runx2 expression in bone and osteoblasts (p<0.05). CONCLUSIONS/SIGNIFICANCE: These results suggest SPI and E2 have different effects on bone turnover prior to puberty. Approximately half of the genes are regulated in the same direction by E2 or SPI, but in combination, SPI blocks the estrogen effects and returns the profile towards control levels. In addition, there are E2 specific and SPI-specific gene changes related to regulation of bone formation.

Feeding blueberry diets in early life prevent senescence of osteoblasts and bone loss in ovariectomized adult female rats.

BACKGROUND: Appropriate nutrition during early development is essential for maximal bone mass accretion; however, linkage between early nutrition, childhood bone mass, peak bone mass in adulthood, and prevention of bone loss later in life has not been studied. METHODOLOGY AND PRINCIPAL FINDINGS: In this report, we show that feeding a high quality diet supplemented with blueberries (BB) to pre-pubertal rats throughout development or only between postnatal day 20 (PND20) and PND34 prevented ovariectomy (OVX)-induced bone loss in adult life. This protective effect of BB is due to suppression of osteoblastic cell senescence associated with acute loss of myosin expression after OVX. Early exposure of pre-osteoblasts to serum from BB-fed rats was found to consistently increase myosin expression. This led to maintenance osteoblastic cell development and differentiation and delay of cellular entrance into senescence through regulation of the Runx2 gene. High bone turnover after OVX results in insufficient collagenous matrix support for new osteoblasts and their precursors to express myosin and other cytoskeletal elements required for osteoblast activity and differentiation. CONCLUSIONS/SIGNIFICANCE: These results indicate: 1) a significant prevention of OVX-induced bone loss from adult rats can occur with only 14 days consumption of a BB-containing diet immediately prior to puberty; and 2) the molecular mechanisms underlying these effects involves increased myosin production which stimulates osteoblast differentiation and reduces mesenchymal stromal cell senescence.

Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats.

Previous in vitro data suggest that ethanol (EtOH) activates NADPH oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption observed in vivo after EtOH exposure. In a rat model in which cycling females were infused intragastrically with EtOH-containing liquid diets, EtOH significantly decreased bone formation and stimulated osteoblast-dependent osteoclast differentiation. These effects were reversed by exogenous 17-ß-estradiol coadministration. Moreover, coadministration of N-acetyl cysteine (NAC), an antioxidant, or diphenylene iodonium (DPI), a specific Nox inhibitor, also abolished chronic EtOH-associated bone loss. EtOH treatment up-regulated mRNA levels of Nox1, 2, 4, and the receptor activator of nuclear factor-κB ligand (RANKL), an essential factor for differentiation of osteoclasts in bone. Protein levels of Nox4, a major Nox isoform expressed in nonphagocytic cells, was also up-regulated by EtOH in bone. 17-ß-Estradiol, NAC, and DPI were able to normalize EtOH-induced up-regulation of Nox and RANKL. In vitro experiments demonstrated that EtOH directly up-regulated Nox expression in osteoblasts. Pretreatment of osteoblasts with DPI eliminated EtOH-induced RANKL promoter activity. Furthermore, EtOH induced RANKL gene expression, and RANKL promoter activation in osteoblasts was ROS-dependent. These data suggest that inhibition of Nox expression and activity may be critical for prevention of chronic EtOH-induced osteoblast-dependent bone loss.

Dietary-induced serum phenolic acids promote bone growth via p38 MAPK/ß-catenin canonical Wnt signaling.

Diet and nutritional status are critical factors that influences bone development. In this report we demonstrate that a mixture of phenolic acids found in the serum of young rats fed blueberries (BB) significantly stimulated osteoblast differentiation, resulting in significantly increased bone mass. Greater bone formation in BB diet-fed animals was associated with increases in osteoblast progenitors and osteoblast differentiation and reduced osteoclastogenesis. Blockade of p38 phosphorylation eliminated effects of BB on activation of Wnt signaling in preosteoblasts. Knocking down ß-catenin expression also blocked the ability of serum from BB diet-fed rats to stimulate osteoblast differentiation in vitro. Based on our in vivo and in vitro data, we propose that the underlying mechanisms of these powerful bone-promoting effects occur through ß-catenin activation and the nuclear accumulation and transactivation of TCF/LEF gene transcription in bone and in osteoblasts. These results indicate stimulation of molecular events leading to osteoblast differentiation triggered by P38 MAP kinase (MAPK)/ß-catenin canonical Wnt signaling results in significant increases in bone growth in young rats consuming BB-supplemented diets. Liquid chromatography/mass spectrometry (LC/MS) characterization of the serum after BB feeding revealed a mixture of simple phenolic acids that may provide a basis for developing a new treatment to increase peak bone mass and delay degenerative bone disorders such as osteoporosis.

A role for ethanol-induced oxidative stress in controlling lineage commitment of mesenchymal stromal cells through inhibition of Wnt/beta-catenin signaling.

The mechanisms by which chronic ethanol intake induces bone loss remain unclear. In females, the skeletal response to ethanol varies depending on physiologic status (e.g., cycling, pregnancy, or lactation). Ethanol-induced oxidative stress appears to be a key event leading to skeletal toxicity. In this study, ethanol-containing liquid diets were fed to postlactational female Sprague-Dawley rats intragastrically for 4 weeks beginning at weaning. Ethanol consumption decreased bone mineral density (BMD) compared with control animals during this period of bone rebuilding following the end of lactation. Coadministration of the antioxidant N-acetylcysteine (NAC) was able to block bone loss and downregulation of the bone-formation markers alkaline phosphatase and osteocalcin in serum and gene expression in bone. Real-time array analysis of total RNA isolated from bone tissue revealed that the majority of Wnt signaling components were downregulated by chronic ethanol infusion. Real-time PCR confirmed downregulated gene expression in a subset of the Wnt signaling components by ethanol. However, the Wnt antagonist DKK1 was upregulated by ethanol. The key canonical Wnt signaling molecule beta-catenin protein expression was inhibited, while glycogen synthase kinase-3-beta was dephosphorylated by ethanol in bone and preosteoblastic cells. These actions of ethanol were blocked by NAC. Ethanol treatment inactivated TCF/LEF gene transcription, eliminated beta-catenin nuclear translocation in osteoblasts, and reciprocally suppressed osteoblastogenesis and enhanced adipogenesis. These effects of ethanol on lineage commitment of mesenchymal stem cells were eliminated by NAC pretreatment. These observations are consistent with the hypothesis that ethanol inhibits bone formation through stimulation of oxidative stress to suppress Wnt signaling.

PPARgamma2 nuclear receptor controls multiple regulatory pathways of osteoblast differentiation from marrow mesenchymal stem cells.

Rosiglitazone (Rosi), a member of the thiazolidinedione class of drugs used to treat type 2 diabetes, activates the adipocyte-specific transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). This activation causes bone loss in animals and humans, at least in part due to suppression of osteoblast differentiation from marrow mesenchymal stem cells (MSC). In order to identify mechanisms by which PPARgamma2 suppresses osteoblastogenesis and promotes adipogenesis in MSC, we have analyzed the PPARgamma2 transcriptome in response to Rosi. A total of 4,252 transcriptional changes resulted when Rosi (1 microM) was applied to the U-33 marrow stromal cell line stably transfected with PPARgamma2 (U-33/gamma2) as compared to non-induced U-33/gamma2 cells. Differences between U-33/gamma2 and U-33 cells stably transfected with empty vector (U-33/c) comprised 7,928 transcriptional changes, independent of Rosi. Cell type-, time- and treatment-specific gene clustering uncovered distinct patterns of PPARgamma2 transcriptional control of MSC lineage commitment. The earliest changes accompanying Rosi activation of PPARgamma2 included effects on Wnt, TGFbeta/BMP and G-protein signaling activities, as well as sustained induction of adipocyte-specific gene expression and lipid metabolism. While suppression of osteoblast phenotype is initiated by a diminished expression of osteoblast-specific signaling pathways, induction of the adipocyte phenotype is initiated by adipocyte-specific transcriptional regulators. This indicates that distinct mechanisms govern the repression of osteogenesis and the stimulation of adipogenesis. The co-expression patterns found here indicate that PPARgamma2 has a dominant role in controlling osteoblast differentiation and suggests numerous gene-gene interactions that could lead to the identification of a "master" regulatory scheme directing this process.

Ethanol impairs estrogen receptor signaling resulting in accelerated activation of senescence pathways, whereas estradiol attenuates the effects of ethanol in osteoblasts.

Epidemiological and animal studies have suggested that chronic alcohol consumption is a major risk factor for osteoporosis. Using bone from cycling female rats infused chronically with ethanol (EtOH) in vivo and osteoblastic cells in vitro, we found that EtOH significantly increased estrogen receptor alpha (ERalpha) and beta (ERbeta) mRNA and ERalpha protein levels. Treatment with 17beta-estradiol (E2) in vivo and in vitro interfered with these effects of EtOH on bone and osteoblastic cells. ERalpha agonist propylpyrazoletriol (PPT) and ERbeta agonist diarylpropionitrile (DPN) attenuated EtOH-induced ERalpha and ERbeta gene overexpression, respectively. Similar to the ER antagonist ICI 182780, EtOH blocked nuclear translocation of ERalpha-ECFP in the presence of E2 in UMR-106 osteoblastic cells. EtOH also downregulated ERE-luc reporter activity. On the other hand, EtOH by itself upregulated some common ERalpha- and ERbeta-mediated genes apparently by an ER-independent pathway. EtOH also transactivated the luciferase activity of the p21 promoter region independent of additional exogenous ERalpha, activated p21 and p53, and stimulated senescence-associated beta-galactosidase activity in rat stromal osteoblasts. E2 treatment attenuated these EtOH actions. We conclude that inhibitory cross-talk between EtOH and E2 in osteoblasts on ERs, p53/p21, and cell senescence provides a pathophysiologic mechanism underlying bone loss and the protective effects of estrogens in alcohol-exposed females.