Protective Effects of Estradiol on Ethanol-Induced Bone Loss Involve Inhibition of Reactive Oxygen Species Generation in Osteoblasts and Downstream Activation of the Extracellular Signal-Regulated Kinase/Signal Transducer and Activator of Transcription 3/Receptor Activator of Nuclear Factor- B Li... (original) (raw)
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Inhibition of NADPH Oxidases Prevents Chronic Ethanol-Induced Bone Loss in Female Rats
Journal of Pharmacology and Experimental Therapeutics, 2011
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.
Journal of Bone and Mineral Research, 2009
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.
Journal of Bone and Mineral Research, 2009
Estrogens diminish oxidative stress in bone and bone marrow, attenuate the generation of osteoblasts, and decrease the prevalence of mature osteoblast apoptosis. We have searched for the molecular mechanism of these effects using as tools a mouse model bearing an estrogen receptor a (ERa) knock-in mutation that prevents binding to DNA (ERa NERKI/À ) and several osteoblast progenitor cell models expressing the wild-type ERa or the ERa NERKI/À . We report that the ability of estrogens to diminish the generation of reactive oxygen species, stimulate the activity of glutathione reductase, and decrease the phosphorylation of p66 shc , as well as osteoblastogenesis and osteoblast number and apoptosis, were fully preserved in ERa NERKI/À mice, indicating that the DNA-binding function of the ERa is dispensable for all these effects. Consistent with the attenuation of osteoblastogenesis in this animal model, 17b-estradiol attenuated bone morphogenetic protein 2 (BMP-2)-induced gene transcription and osteoblast commitment and differentiation in murine and human osteoblastic cell lines. Moreover, 17b-estradiol attenuated BMP-2-induced differentiation of primary cultures of calvaria-or bone marrow-derived osteoblastic cells from ERa NERKI/À mice as effectively as in cells from wild-type littermates. The inhibitory effect of the hormone on BMP-2 signaling resulted from an ERa-mediated activation of ERKs and the phosphorylation of Smad1 at the linker region of the protein, which leads to proteasomal degradation. These results illustrate that the effects of estrogens on oxidative stress and the birth and death of osteoblasts do not require the binding of ERa to DNA response elements, but instead they result from the activation of cytoplasmic kinases. ß 2010 American Society for Bone and Mineral Research.
Alcoholism: Clinical and Experimental Research, 2016
Background-Chronic alcohol consumption leads to increased fracture risk and an elevated risk of osteoporosis by decreasing bone accrual through increasing osteoclast activity and decreasing osteoblast activity. We have shown that this mechanism involves the generation of reactive oxygen species (ROS) produced by NADPH oxidases (NOX). It was hypothesized that different dietary antioxidants, N-acetyl cysteine (NAC, 1.2mg/kg/d) and α-tocopherol (VitE, 60 mg/kg/d)) would be able to attenuate the NOX-mediated ROS effects on bone due to chronic alcohol intake. Methods-To study the effects of these antioxidants, female mice received a Lieber DeCarli liquid diet containing ethanol (EtOH) with or without additional antioxidant for 8 weeks. Results-Tibias displayed decreased cortical bone mineral density in both the EtOH and EtOH +antioxidant groups compared to pair-fed (PF) and PF+antioxidant groups (P<0.05). However, there was significant protection from trabecular bone loss in mice fed either antioxidant (P<0.05). MicroCT analysis demonstrated a significant decrease in bone volume (BV/TV) and trabecular number (Tb.N) (P<0.05), along with a significant increase in trabecular spacing (Tb.Sp) in the EtOH compared to PF (P<0.05). In contrast, the EtOH+NAC and EtOH+α-tocopherol did not statistically differ from their respective PF controls. Ex vivo histological sections of tibias were stained for nitrotyrosine, an indicator of intracellular damage by ROS, and tibias from mice fed EtOH exhibited significantly more staining than PF controls. EtOH treatment significantly
Different Molecular Mechanisms Underlie Ethanol-Induced Bone Loss in Cycling and Pregnant Rats
Endocrinology, 2006
nia. In the current study, we examined the modulation of EtOH-induced bone loss during pregnancy. Nonpregnant and pregnant dams were intragastrically infused either control or EtOH-containing diets throughout gestation (gestation d 5 through 20 or an equivalent period of 15 d) by total enteral nutrition. The effects of EtOH (8.5 to 14 g/kg/d) on tibial bone mineral density (BMD), mineral content (BMC), and bone mineral area were assessed at gestation d 20 via peripheral quantitative computerized tomography. EtOH caused a dose-dependent decrease in BMD and BMC without affecting bone mineral area. Trabecular BMD and BMC were significantly lower in EtOH-treated, nonpregnant dams, compared with pregnant cohorts at the same infused dose of EtOH and urinary ethanol concentrations. Static histomorphometric analysis of tibiae from pregnant rats after EtOH treatment showed decreased osteoblast and osteoid surface, indicating inhibited bone formation, whereas EtOH-treated cycling rats showed higher osteoclast and eroded surface, indicative of increased bone resorption. Circulating osteocalcin and 1,25-dihydroxyvitamin D 3 were lower in both EtOH-fed nonpregnant and pregnant rats. Gene expression of osteoclast markers, 70 kDa v-ATPase, and tartrate-resistant acid phosphatase were increased selectively in nonpregnant EtOH-treated rats but not pregnant rats. Moreover, only nonpregnant EtOH-fed rats showed induction in bone marrow receptor activator of nuclear factor-B ligand mRNA and decreased circulating 17estradiol levels. Our data suggest that EtOH-induced bone loss in pregnant rats is mainly due to inhibited bone formation, whereas in nonpregnant rats, the data are consistent with increased osteoclast activation and bone resorption concomitant with decreased estradiol levels.
Toxicology, 2007
With rising rates of alcohol consumption acute and chronic damage from alcohol is expected to increase all over the world. Habitual excessive alcohol consumption is associated with pathological effects on bone. The aim of the present in vitro study was to investigate comparatively the proliferation and synthetic activity of osteoblasts (OB) isolated from the trabecular bone of rats previously exposed to 7-week intermittent exposure to ethanol vapor, sham-aged rats and long-term estrogen deficient rats. Cell proliferation (WST1) and synthesis of alkaline phosphatase (ALP), osteocalcin (OC), collagen I (CICP), transforming growth factor beta1 (TGF-1), interleukin-6 (IL-6), tumor necrosis factor alfa (TNF␣) were measured at 3, 7 and 14 days of culture.
Molecular and Cellular Biology - MOL CELL BIOL, 2007
Estrogens control gene transcription by cis or trans interactions of their receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-Estren-3α, 17β-diol (estren) or an estradiol-dendrimer conjugate (EDC), two synthetic compounds that stimulate kinase-mediated ER actions 1,000 -10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17β-estradiol (E 2 ), suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E 2 , stimulated Wnt/βcatenin-mediated transcription in TCF-lacZ transgenic mice. Moreover, E 2 stimulated BMP signaling in mice in which ERα lacks DNA binding activity and classical ERE-mediated transcription (ERα NERKI/-), but not in wild type controls. This evidence reveals for the first time the existence of a large signalosome, in which inputs from the ER, kinases, BMPs, and Wntsignaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.
The Journal of pharmacology and experimental therapeutics, 2018
Chronic alcohol consumption increases bone resorption and decreases bone formation. A major component of ethanol (EtOH) pathology in bone is the generation of excess reactive oxygen species (ROS). The ROS-generating NADPH oxidase-4 (NOX4) is proposed to drive much of the EtOH-induced suppression of bone formation. Here, 13-week-old male wild-type (WT) and NOX4 mice were pair fed (PF) a high-fat (35%), Lieber-DeCarli liquid diet with or without EtOH at 30% of their total calories for 12 weeks. Micro-computed tomography analysis demonstrated significant decreases in trabecular bone volume/total volume (BV/TV) percentage and cortical thickness in WT, EtOH-fed mice compared with PF controls. EtOH-fed NOX4 mice also displayed decreased trabecular BV/TV and trabecular number compared with PF ( < 0.05). However, NOX4 mice were protected against EtOH-induced decreases in cortical thickness ( < 0.05) and decreases in collagen1 and osteocalcin mRNA expression in cortical bone ( < 0.0...
Role of estrone on the regulation of osteoblastogenesis
Molecular and Cellular Endocrinology, 2019
Although estradiol bone contribution has been deeply studied, little is known about the action of estrone. We investigated the direct action of estrone on osteoblasts growth and differentiation, with focus on the biochemical mechanism displayed by the estrogen. Murine calvarial osteoblast cultures in vitro exposed to 10 nM estrone were employed. Estrone enhanced gene expression of the osteogenic differentiation marker, Runx2 mRNA (150% above control). The hormone significantly increased cell proliferation (38% above control), nitric oxide production (108% above control), alkaline phosphatase activity (50% above control), in addition to stimulation of extracellular matrix mineralization. Using specific antagonists, we found that the mechanism of action of estrone involves estrogen receptor, nitric oxide synthase and MAPK signalling pathways participation. The hormone acts by its own and probably not via conversion to estradiol, since 17 B HSD inhibition did not affect the hormonal action. This work shows a novel action of estrone on bone cells promoting osteoblastogenesis.