Abstract
Bone loss occurs following chronic ethanol (EtOH) consumption in males and cycling females in part as a result of increased bone resorption. We have demonstrated in vivo that estradiol treatment can reverse this effect. Using osteoclast precursors from bone marrow and osteoblast/preosteoclast coculture, we found that EtOH-induced receptor activator of nuclear factor-κB ligand (RANKL) expression in osteoblasts was able to promote osteoclastogenesis. These effects were blocked by pretreatment of cells with either 17β-estradiol (E2) or the anti-oxidant N-acetyl cysteine (NAC). EtOH treatment of stromal osteoblasts increased the intracellular level of reactive oxygen species (ROS). This was associated with induction of NADPH oxidase (NOX) and a downstream signaling cascade involving sustained activation of extracellular signal-regulated kinase (ERK) and activation of signal transducer and activator of transcription 3, resulting in increased gene expression of RANKL. In the presence of EtOH, sustained nuclear ERK translocation >24 h was observed in calvarial osteoblasts and UMR-106 cells transfected with green fluorescent protein-ERK2 plasmid. This was abolished by pretreatment with either E2 or NAC. NOX subtypes 1, 2, and 4, but not 3, were expressed in stromal osteoblasts. Chemical inhibition of NOX by diphenylene iodonium also reversed the ability of EtOH to phosphorylate ERK and induce RANKL mRNA expression. Down-regulation of EtOH-induced ROS generation in osteoblasts was also observed after treatment with E2 or NAC. These data suggest that the molecular mechanisms whereby E2 prevents EtOH-induced bone loss involve interference with ROS generation and cytoplasmic kinase activation.
Footnotes
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This study was supported by in part by National Institutes of Health Grant R01 AA12928 (to M.J.J.R.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.130351.
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ABBREVIATIONS: EtOH, ethanol; RANKL, receptor activator of nuclear factor-κB ligand; ADH, alcohol dehydrogenase; ROS, reactive oxygen species; NOX, nicotinamide adenine dinucleotide phosphate oxidase; ERK, extracellular signal-regulated kinase; E2, 17-β-estradiol; STAT, signal transducer and activator of transcription; FBS, fetal bovine serum; TRAPase, tartrate-resistant acid phosphatase; RT-PCR, reverse transcription-polymerase chain reaction; Chx, cycloheximide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 2,7-DCF-DA, 2,7-dichlorodihydrofluorescein diacetate; NAC, N-acetyl cysteine; GFP, green fluorescent protein; MEK, mitogen-activated protein kinase kinase; nRFP, nuclear red fusion protein; ANOVA, analysis of variance; DPI, diphenylene iodonium; P-, phosphorylated; T-, total.
- Received August 17, 2007.
- Accepted October 2, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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