Although it is well known that women are more susceptible to the toxic effects of ethanol (EtOH) than men, much less is known about the molecular mechanisms underlying alcohol toxicity in women especially as relates to bone. Alcohol abuse during early adulthood results in impaired bone growth and in the U.S.A. approximately 20% of women age 18-30 (4.4 million) binge drink. A resulting reduction in peak bone mass would predispose women to osteoporosis in later life. The molecular mechanisms underlying the toxic effects of EtOH on bone remain poorly understood. EtOH, particularly at higher concentrations characteristic of binge drinking, can increase bone resorption. We have developed a new mouse model to study alcohol-induced bone loss in cycling females. Chronic consumption of EtOH as part of liquid diets using this system produces EtOH-induced bone loss without compromising food intake. Bone loss in female mice was accompanied by increased serum markers of bone resorption and increased osteoclast numbers in ex-vivo bone marrow cultures. These data are consistent with previously published data from our laboratory in cycling female rats fed ethanol via total enteral nutrition. In the rat model, increased bone resorption was accompanied by increased expression of RANKL in bone. RANKL is a member of the TNF family expressed on the surface of osteoblasts which signals through the receptor RANK on the surface of osteoclast precursors to stimulate osteoclastogenesis. EtOH-induction of RANKL occurred in primary osteoblast cultures from rats and mice and in rat and mouse UMR-106 and ST-2 osteoblast-like cell lines. EtOH-induction of RANKL was blocked by the antioxidant N-acetylcysteine (NAC) in vitro. RANKL appears to be regulated via ERK1/2 and STAT3 phosphorylation. In vitro data suggest that the ERK/STAT3/RANKL pathway is stimulated by reactive oxygen species (ROS) produced by an ethanol-induced NADPH oxidase (NOX4). Expression of this enzyme in osteoblasts is increased by EtOH and blocked by NAC. The mouse liquid diet model will be utilized to determine the role of NOX4 and oxidative stress in EtOH-induced bone resorption in vivo using p47phox -/- mice and catalase transgenic mice. In vitro, the mouse ST-2 cell line, differentiated mouse stromal osteoblast cultures and osteoblast/osteoclast precursor co-cultures will be utilized to probe the molecular cascade from EtOH to RANKL and the inhibition of EtOH-induced RANKL by NAC and other antioxidants. NOX gain and loss of function studies will be performed by transient transfection and use of siRNA and the effects of hydrogen peroxide and free radical scavenging on the ERK/STAT3/RANKL cascade will be examined. In addition, in vivo studies will be conducted to examine the ability of antioxidants to block EtOH-induced bone resorption.

Public Health Relevance

Excessive alcohol consumption is an increasing problem in young women. The results from research proposed herein will link the deleterious effects of alcohol consumption on bone in women of reproductive age with osteoporosis, a major cause of morbidity and mortality in postmenopausal women. Alcohol abuse results in osteopenia and increases osteoporosis risk. One potentially important yet under-explored area of bone health is the relationship between bone resorption associated with alcohol abuse in young women and increased risk of osteoporosis which results from impairments in attainment of peak bone mass. In addition, alcohol-induced bone loss shares many features in common with bone loss during menopause and aging. Our animal data suggests that dietary antioxidants may prevent alcohol-induced bone resorption and we will test this in the current application. Molecular studies of alcohol actions on ROS signaling cascades in bone are expected to provide fundamental insights into common pathways underlying bone resorption.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Research Project (R01)
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Special Emphasis Panel (ZRG1-DKUS-C (90))
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Orosz, Andras
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Arkansas Children's Hospital Research Institute
Little Rock
United States
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Alund, Alexander W; Mercer, Kelly E; Suva, Larry J et al. (2016) Reactive Oxygen Species Differentially Regulate Bone Turnover in an Age-Specific Manner in Catalase Transgenic Female Mice. J Pharmacol Exp Ther 358:50-60
Chen, Jin-Ran; Lazarenko, Oxana P; Blackburn, Michael L et al. (2015) p47phox-Nox2-dependent ROS Signaling Inhibits Early Bone Development in Mice but Protects against Skeletal Aging. J Biol Chem 290:14692-704
Mercer, Kelly E; Hennings, Leah; Sharma, Neha et al. (2014) Alcohol consumption promotes diethylnitrosamine-induced hepatocarcinogenesis in male mice through activation of the Wnt/?-catenin signaling pathway. Cancer Prev Res (Phila) 7:675-85
Mercer, Kelly E; Sims, Clark R; Yang, Carrie S et al. (2014) Loss of functional NADPH oxidase 2 protects against alcohol-induced bone resorption in female p47phox-/- mice. Alcohol Clin Exp Res 38:672-82
Ronis, Martin J J; Mercer, Kelly; Suva, Larry J et al. (2014) Influence of fat/carbohydrate ratio on progression of fatty liver disease and on development of osteopenia in male rats fed alcohol via total enteral nutrition (TEN). Alcohol 48:133-44
Yang, Carrie S; Mercer, Kelly E; Alund, Alexander W et al. (2014) Genistein supplementation increases bone turnover but does not prevent alcohol-induced bone loss in male mice. Exp Biol Med (Maywood) 239:1380-9
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Mercer, Kelly E; Wynne, Rebecca A; Lazarenko, Oxana P et al. (2012) Vitamin D supplementation protects against bone loss associated with chronic alcohol administration in female mice. J Pharmacol Exp Ther 343:401-12
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