Estradiol protects against ethanol-induced bone loss by inhibiting up-regulation of receptor activator of nuclear factor-κB ligand in osteoblasts

To investigate the effects of sex hormones on ethanol (EtOH)induced bone loss, female Sprague-Dawley rats were fed control or EtOH-containing diets (12 g/kg/day) by intragastric infusion. After 3 weeks, rats receiving EtOH had significant decreases in tibial trabecular and total bone mineral density, induction of receptor activator of nuclear factor-kappa B ligand (RANKL) mRNA expression, and enhanced bone resorption, all of which were prevented by treatment with 17 beta-estradiol (E-2). The addition of progesterone did not enhance the beneficial effect of E 2 alone. Consistent with our in vivo findings, EtOH stimulated RANKL mRNA expression in cultured primary osteoblasts, and this expression was blocked by 4-methylpyrazole. Acetaldehyde also induced RANKL expression. Class 1 alcohol dehydrogenase was found to be expressed and EtOH-inducible in cultured osteoblasts, whereas CYP2E1 was undetectable. We found that EtOH induced phosphorylation of extracellular signal-regulated kinase (ERK) and signal transducers and activators of transcription 3 (STAT3). E-2 and the mitogen-activated protein kinase kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) blocked ERK and STAT3 phosphorylation and blocked RANKL induction. Moreover, E-2 completely blocked EtOH-induced osteoclastogenesis in a primary osteoblast and osteoclast precursor coculture system. The E-2 effects were estrogen receptor-mediated. Therefore, E-2 prevents EtOH-induced bone loss by opposing the induction of RANKL mRNA in osteoblasts and ethanol-induced osteoclastogenesis, through opposing effects on sustained ERK signaling.