Oxana P. Lazarenko

PPARγ2 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 (PPARγ). 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 PPARγ2 suppresses osteoblastogenesis and promotes adipogenesis in MSC, we have analyzed the PPARγ2 transcriptome in response to Rosi. A total of 4,252 transcriptional changes resulted when Rosi (1 µM) was applied to the U‐33 marrow stromal cell line stably transfected with PPARγ2 (U‐33/γ2) as compared to non‐induced U‐33/γ2 cells. Differences between U‐33/γ2 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 PPARγ2 transcriptional control of MSC lineage commitment. The earliest changes accompanying Rosi activation of PPARγ2 included effects on Wnt, TGFβ/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 PPARγ2 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. J. Cell. Biochem. 106: 232–246, 2009.

Obesity Reduces Bone Density Associated with Activation of PPARγ and Suppression of Wnt/β-Catenin in Rapidly Growing Male Rats

Background It 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 Findings Total 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/Significance These 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.

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

Dietary-induced serum phenolic acids promote bone growth via p38 MAPK/β-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 β‐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 β‐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)/β‐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.

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 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.

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-β-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-β-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.

Infant Formula Promotes Bone Growth in Neonatal Piglets by Enhancing Osteoblastogenesis through Bone Morphogenic Protein Signaling

Relatively few studies have examined the effects of formula feeding relative to breast-feeding on bone in the neonate. Using peripheral quantitative CT scan and histomorphometric analysis, we demonstrated that neonatal piglets fed with soy-based formula (SF) and cow milk-based formula (MF) for 21 or 35 d had greater bone mineral density and content than breast-fed piglets (BF) (P < 0.05). Osteoblast numbers and bone formation rate at postnatal d 35 were greater in SF compared with other groups (P < 0.05), whereas osteoclast numbers were lower in both MF and SF groups than in the BF group (P < 0.05). Osteoblastogenesis was greater in ex vivo bone marrow cell cultures from SF than in MF or BF piglets (P < 0.05). Bone formation markers in serum were greater, whereas bone resorption markers were lower in the MF- and SF-fed groups than in the BF group (P < 0.05). Bone morphogenic protein (BMP) 2 and alkaline phosphatase mRNAs were upregulated in the MF and SF groups compared with the BF group (P < 0.05), whereas receptor activator of NF-kappaB ligand was downregulated (P < 0.05). Extracellular signal-regulated kinase, p38, Smad1/5/8 phosphorylation, and runt-related transcription factor 2 expression were greater in bone from the MF and SF groups compared with the BF group (P < 0.05). In vitro studies showed that 2.5% serum from SF- or MF-fed piglets was able to stimulate osteoblast differentiation but not in the presence of the BMP blocker noggin. Therefore, formula feeding promoted bone growth compared with BF. SF piglets had the highest bone volume over tissue volume. This suggests that SF-fed piglets may have the best quality bone. The anabolic effects of SF on bone appear to be mediated through enhanced BMP signaling.

Inhibition of fetal bone development through epigenetic down-regulation of HoxA10 in obese rats fed high-fat diet

Epidemiological studies show that maternal obesity during intrauterine and early postnatal life increases the risk of low bone mass and fracture later in life. Here, we show that bone development is inhibited in gestational embryonic day 18.5 (E18.5) embryos from rat dams made obese by feeding a high‐fat diet (HFD). Moreover, fetal rat osteogenic calvarial cells (FOCCs) from these obese dams have significantly less potential to develop into mature osteoblasts compared to cells from AIN‐93G diet‐fed controls. Profiling of transcriptional genes for osteogenesis revealed a profound decrease in the homeodomain‐containing factor A10 (HoxA10) in FOCCs from fetuses of HFD‐induced obese dams. Significant methylation of the HoxA10 promoter was found in those FOCCs, as well as in mouse ST2 cells treated with a mixture of free fatty acids similar to that found in serum from HFD‐induced obese rats. This was accompanied by lower expression of osteogenic markers, but higher levels of PPARγ. Control FOCCs depleted of the HoxA10 gene (shRNA) ex vivo behave similarly to cells from fetuses of obese dams; conversely, overexpression of HoxA10 gene in FOCCs from HFD rats exhibit the same phenotype as controls. Treatment of FOCCs from control rats or of ST2 cells with an artificial mixture of free fatty acids significantly down‐regulated HoxA10 protein expression, and cells exhibited adipocyte‐like properties. These results suggest that maternal obesity impairs fetal skeletal development through down‐regulation of the HoxA10 gene, which may lead to an increase in the prevalence of low bone mass in the offspring later in life.—Chen, J.‐R., Zhang, J., Lazarenko, O. P., Kang, P., Blackburn, M. L., Ronis, M. J. J., Badger, T. M., Shankar, K. Inhibition of fetal bone development through epigenetic down‐regulation of HoxA10 in obese rats fed high‐fat diet. FASEB J. 26, 1131‐1141 (2012). www.fasebj.org

Short Term Effects on Bone Quality Associated with Consumption of Soy Protein Isolate and Other Dietary Protein Sources in Rapidly Growing Female Rats

Beneficial effects of soy protein consumption on bone quality have been reported. The effects of other dietary protein sources such as whey protein hydrolysate (WPH) and rice protein isolate (RPI) on bone growth have been less well examined. The current study compared effects of feeding soy protein isolate (SPI), WPH and RPI for 14 d on tibial bone mineral density (BMD) and bone mineral content (BMC) in intact and ovariectomized (OVX) rapidly growing female rats relative to animals fed casein (CAS). The effects of estrogenic status on responses to SPI were also explored. Tibial peripheral quantitative computerized tomography (pQCT) showed all three protein sources had positive effects on either BMD or BMC relative to CAS (P < 0.05), but SPI had greater effects in both intact and OVX female rats. SPI and E2 had positive effects on BMD and BMC in OVX rats (P < 0.05). However, trabecular BMD was lower in a SPI + E2 group compared to a CAS + E2 group. In OVX rats, SPI increased serum bone formation markers, and serum from SPI-fed rats stimulated osteoblastogenesis in ex vivo. SPI also suppressed the bone resorption marker RatLaps (P < 0.05). Both SPI and E2 increased alkaline phosphatase gene expression in bone, but only SPI decreased receptor activator of nuclear factor-κB ligand (RANKL) and estrogen receptor gene expression (P < 0.05). These data suggest beneficial bone effects of a soy diet in rapidly growing animals and the potential for early soy consumption to increase peak bone mass.