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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPACT OF EARLY DIETARY FACTORS ON CHILD DEVELOPMENT AND HEALTH Title: Loss of functional NADPH oxidase-2 protects against alcohol-induced bone resorption in female p47phox-/- mice

Authors
item Mercer, Kelly -
item Sims, Clark -
item Yang, Carrie -
item Wynne, Rebecca -
item Moutos, Christopher -
item Hogue, William -
item Lumpkin, Charles -
item Suva, Larry -
item Chen, Jin-Ran -
item Badger, Thomas
item Ronis, Martin -

Submitted to: Alcoholism: Clinical and Experimental
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 13, 2013
Publication Date: November 20, 2013
Citation: Mercer, K.E., Sims, C.R., Yang, C.S., Wynne, R.A., Moutos, C., Hogue, W.R., Lumpkin, C.K., Suva, L.J., Chen, J., Badger, T.M., Ronis, M.J. 2013. Loss of functional NADPH oxidase-2 protects against alcohol-induced bone resorption in female p47phox-/- mice. Alcoholism: Clinical and Experimental Research. 38(3):672-682.

Interpretive Summary: Chronic alcoholic drinking is a well-described risk factor for increased bone loss over one's lifetime. In previous work, we have shown that chronic feeding of alcohol to rodents increases oxidative stress in the bone, which signals for increased numbers of osteoclasts, bone-eroding cells, and decreased numbers of osteoblast, bone-forming cells, which results in more bone eaten away and less bone added to the skeleton, thus bone loss. A family of enzymes, called NADPH oxidases (Nox), is in part responsible for making the free radicals and hydrogen peroxide in these cells. To identify the Nox enzyme that is involved in alcohol-mediated increase in oxidative stress, we gave alcohol to female mice that do not have a functional Nox2 enzyme (p47phox KO). We found that loss of Nox2 enzymatic activity protected the mice from alcohol-mediated bone loss by blocking the signal for increasing osteoclast number and activity. Interestingly, loss of Nox2 enzymatic activity did not prevent the alcohol-mediated loss in bone-forming cells. Additional experiments in cell lines suggest that another family member, Nox4, may be working independently of Nox2 to signal for alcohol-mediated losses in bone-forming cells, and also working in tandem with Nox2 to signal for alcohol-mediated increases in bone erosion.

Technical Abstract: In bone, oxidant signaling through NADPH oxidase (NOX)-derived reactive oxygen species (ROS) is an important stimulus for osteoclast differentiation and activity. We have previously demonstrated that chronic alcohol abuse produces bone loss through NOX-dependent mechanisms. In the current study, six-week-old female C57BL/6J-Ncf1/p47phox-/- (p47phox KO) and wild-type (WT) mice were pair-fed ethanol (EtOH) diets for 40 days to test if NOX2 is necessary for EtOH-induced bone loss. In WT mice, EtOH treatment significantly reduced bone density and mechanical strength, and significantly increased total osteoclast number and activity. In contrast, in EtOH-treated p47phoxKO mice, bone density and mechanical strength were completely preserved. Moreover, we observed no changes in osteoclast numbers or activity, and no elevations in serum CTX or gene expression of the NF'B receptor ligand (RANKL) gene expression in bone. However, in both WT and p47phox KO mice, EtOH-feeding significantly reduced biochemical markers of bone formation. In stromal ST2 cells, in vitro EtOH exposure resulted in an increase in hydrogen peroxide, which was blocked by pre-treating cells with the NOX inhibitors DPI or gliotoxin, but not plumbagin. Longer EtOH exposures produced significant increases in RANKL and NOX2 gene expression. Moreover, pre-incubations of cells with 4-methylpyrazole or plumbagin significantly reduced EtOH-induced RANKL expression. These data suggest that NOX2-derived ROS is necessary for EtOH-induced bone resorption. In osteoblasts, NOX2 and NOX4 appear to work in tandem to increase RANKL expression, whereas EtOH-mediated inhibition of bone formation occurs via a NOX2-independent mechanism.

Last Modified: 10/30/2014
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