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-〉 ligand (RANKL) mRNA expression, and enhanced bone resorption, all of which were prevented by treatment with 17␤-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 EtOHinducible 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 mitogenactivated protein kinase kinase inhibitor 2Ј-amino-3Ј-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.

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.

Chronic ethanol (EtOH) consumption can result in osteopenia. 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 D3 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-kappaB ligand mRNA and decreased circulating 17beta-estradiol 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.