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-kappaB ligand signaling cascade.
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-kappaB ligand (RANKL) expression in osteoblasts was able to promote osteoclastogenesis. These effects were blocked by pretreatment of cells with either 17beta-estradiol (E(2)) 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 E(2) 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 E(2) or NAC. These data suggest that the molecular mechanisms whereby E(2) prevents EtOH-induced bone loss involve interference with ROS generation and cytoplasmic kinase activation.
Chen
Jin-Ran
JR
Arkansas Children's Nutrition Center, Slot 512-20B, 1120 Marshall St., Little Rock, AR 72202, USA.
Shankar
Kartik
K
Nagarajan
Shanmugam
S
Badger
Thomas M
TM
Ronis
Martin J J
MJ
D000111:Acetylcysteine / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001862:Bone Resorption / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D004958:Estradiol / Q000494:pharmacology*; D000431:Ethanol / Q000009:adverse effects*; D005260:Female; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D053245:RANK Ligand / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D012886:Skull / Q000166:cytology
D000975:Antioxidants; D053245:RANK Ligand; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D050796:STAT3 Transcription Factor; C494088:Stat3 protein, rat; D000431:Ethanol; D004958:Estradiol; D019255:NADPH Oxidases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000111:Acetylcysteine