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 b-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 b-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)/b-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. ß

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 β-catenin protein expression was inhibited, while glycogen synthase kinase-3-β 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 β-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. © 2010 American Society for Bone and Mineral Research.

BackgroundIt is well established that excessive consumption of a high fat diet (HFD) results in obesity; however, the consequences of obesity on postnatal skeletal development have not been well studied.Methodology and Principal FindingsTotal enteral nutrition (TEN) was used to feed postnatal day 27 male rats intragastrically with a high 45% fat diet (HFD) for four weeks to induce obesity. Fat mass was increased compared to rats fed TEN diets containing 25% fat (medium fat diet, MFD) or a chow diet (low fat diet, LFD) fed ad libitum with matched body weight gains. Serum leptin and total non-esterified fatty acids (NEFA) were elevated in HFD rats, which also had reduced bone mass compared to LFD-fed animals. This was accompanied by decreases in bone formation, but increases in the bone resorption. Bone marrow adiposity and expression of adipogenic genes, PPARγ and aP2 were increased, whereas osteoblastogenic markers osteocalcin and Runx2 were decreased, in bone in HFD rats compared to LFD controls. The diversion of stromal cell differentiation in response to HFD stemmed from down-regulation of the key canonical Wnt signaling molecule β-catenin protein and reciprocal up-regulation of nuclear PPARγ expression in bone. In a set of in vitro studies using pluripotent ST2 bone marrow mesenchymal stromal cells treated with serum from rats on the different diets or using the free fatty acid composition of NEFA quantified in rat serum from HFD-fed animals by GC-MS, we were able to recapitulate our in vivo findings.Conclusions/SignificanceThese observations strongly suggest that increased NEFA in serum from rats made obese by HFD-feeding impaired bone formation due to stimulation of bone marrow adipogenesis. These effects of obesity on bone in early life may result in impaired attainment of peak bone mass and therefore increase the prevalence of osteoporosis later on in life.

Covalent modifications of histone tails play important roles in gene transcription and silencing. We recently identified an ERG ( ets -related gene)-associated protein with a SET (suppressor of variegation, enhancer of zest and trithorax) domain (ESET) that was found to have the activity of a histone H3-specific methyltransferase. In the present study, we investigated the interaction of ESET with other chromatin remodelling factors. We show that ESET histone methyltransferase associates with histone deacetylase 1 (HDAC1) and HDAC2, and that ESET also interacts with the transcription co-repressors mSin3A and mSin3B. Deletion analysis of ESET reveals that an N-terminal region containing a tudor domain is responsible for interaction with mSin3A/B and association with HDAC1/2, and that truncation of ESET enhances its binding to mSin3. When bound to a promoter, ESET represses the transcription of a downstream luciferase reporter gene. This repression by ESET is independent of its histone methyltransferase activity, but correlates with its binding to the mSin3 co-repressors. In addition, the repression can be partially reversed by treatment with the HDAC inhibitor trichostatin A. Taken together, these data suggest that ESET histone methyltransferase can form a large, multi-protein complex(es) with mSin3A/B co-repressors and HDAC1/2 that participates in multiple pathways of transcriptional repression.

Maternal obesity at conception increases the risk of offspring obesity, thus propagating an intergenerational vicious cycle. Male offspring born to obese dams are hyperresponsive to high fat-diets, gaining greater body weight, fat mass, and additional metabolic sequelae compared to lean controls. In this report, we identify the impact of maternal obesity before conception, on the embryo, and intrauterine milieu during the periimplantation period. We conducted global transcriptomic profiling in the uterus and periimplantation blastocyst, gene/protein expression analyses of inflammatory pathways in conjunction with endocrine and metabolic characterization in the dams at implantation. Uterine gene expression profiles of lean and obese dams revealed distinct signatures for genes regulating inflammation and lipid metabolism. Both pathway and gene-set enrichment analysis revealed uterine nuclear factor-κB and c-Jun N-terminal kinase signaling to be up-regulated in the uterus of obese dams, which was confirmed via immunoblotting. Obese uteri also evidenced an inflammatory secretome with higher chemokine mRNA abundance (CCL2, CCL5, CCL7, and CxCL10) and related regulators (TLR2, CD14, and Ccr1). Increased inflammation in the uterus was associated with ectopic lipid accumulation and expression of lipid metabolic genes. Gene expression in sex-identified male periimplantation blastocyst at day postcoitum 4.5 was clearly influenced by maternal obesity (359 transcripts, ±1.4-fold), including changes in developmental and epigenetic regulators. Akin to the uterus, nuclear factor-κB-regulated proinflammatory genes (CCL4 and CCL5) increased and expression of antioxidant (GPx3) and mitochondrial (TFAM and NRF1) genes decreased in the obese embryos. Our results suggest that ectopic lipid and inflammation may link maternal obesity to increased predisposition of offspring to obesity later in life.

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 α (ERα) and β (ERβ) mRNA and ERα protein levels. Treatment with 17β-estradiol (E2) in vivo and in vitro interfered with these effects of EtOH on bone and osteoblastic cells. ERα agonist propylpyrazoletriol (PPT) and ERβ agonist diarylpropionitrile (DPN) attenuated EtOH-induced ERα and ERβ gene overexpression, respectively. Similar to the ER antagonist ICI 182780, EtOH blocked nuclear translocation of ERα-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 ERα- and ERβ-mediated genes apparently by an ER-independent pathway. EtOH also transactivated the luciferase activity of the p21 promoter region independent of additional exogenous ERα, activated p21 and p53, and stimulated senescence-associated β-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.

In utero exposure to maternal obesity increases the offspring's risk of obesity in later life. We have also previously reported that offspring of obese rat dams develop hepatic steatosis, mild hyperinsulinemia, and a lipogenic gene signature in the liver at postnatal day (PND)21. In the current study, we examined systemic and hepatic adaptations in male Sprague-Dawley offspring from lean and obese dams at PND21. Indirect calorimetry revealed decreases in energy expenditure (p<0.001) and increases in RER values (p<0.001), which were further exacerbated by high fat diet (45% kcals from fat) consumption indicating an impaired ability to utilize fatty acids in offspring of obese dams as analyzed by PRCF. Mitochondrial function is known to be associated with fatty acid oxidation (FAO) in the liver. Several markers of hepatic mitochondrial function were reduced in offspring of obese dams. These included SIRT3 mRNA (p = 0.012) and mitochondrial protein content (p = 0.002), electron transport chain complexes (II, III, and ATPase), and fasting PGC-1α mRNA expression (p<0.001). Moreover, hepatic LCAD, a SIRT3 target, was not only reduced 2-fold (p<0.001) but was also hyperacetylated in offspring of obese dams (p<0.005) suggesting decreased hepatic FAO. In conclusion, exposure to maternal obesity contributes to early perturbations in whole body and liver energy metabolism. Mitochondrial dysfunction may be an underlying event that reduces hepatic fatty acid oxidation and precedes the development of detrimental obesity associated co-morbidities such as insulin resistance and NAFLD.

The dcfining cytogenetic abnormality of Ewing's sarcoma is the presence of a balanced t( 1 1;22) translocation expressing the EWSIFLI-I chimeric fusion protein. The effect of EWSIFLI-1 appears to be dominant negative since over-expression of EWS does not overcome the sarcoma phenotype. Previous studies have shown that EWSIFLI-1 as well as related sarcoma fusion proteins are necessary and sufficient to induce transformation both in vitro and in vivo. In this study we report that synthetic small interfering RNA (siRNA) specifically suppresses EWSIFLI-1 fusion gene expression in SK-ES Ewing's sarcoma cells. Knockdown of the EWSI FLI-1 fusion protein is correlated with decreased cell proliferation and increased apoptosis. We demonstrate that Ewing's sarcoma tumors as well as Ewing's sarcoma cell lines predominantly express the CXCR4 chemokine receptor. Using an in vitro invasion assay, the SDF-1 ligand of CXCR4 was shown to be a potent stimulus of invasion by SK-ES cells. Knockdown of EWSIFLI-1 by RNA interference abrogates the invasiveness of SK-ES cells. These experiments suggest that targeted silencing of the EWSIFLI-I fusion gene. by siRNA represents a promising strategy to study the loss of EWSIFLI-I protein in Ewing's sarcoma cells of otherwise identical genetic background.