Abstract
Previous in vitro data suggest that ethanol (EtOH) activates NADPH oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption observed in vivo after EtOH exposure. In a rat model in which cycling females were infused intragastrically with EtOH-containing liquid diets, EtOH significantly decreased bone formation and stimulated osteoblast-dependent osteoclast differentiation. These effects were reversed by exogenous 17-β-estradiol coadministration. Moreover, coadministration of N-acetyl cysteine (NAC), an antioxidant, or diphenylene iodonium (DPI), a specific Nox inhibitor, also abolished chronic EtOH-associated bone loss. EtOH treatment up-regulated mRNA levels of Nox1, 2, 4, and the receptor activator of nuclear factor-κB ligand (RANKL), an essential factor for differentiation of osteoclasts in bone. Protein levels of Nox4, a major Nox isoform expressed in nonphagocytic cells, was also up-regulated by EtOH in bone. 17-β-Estradiol, NAC, and DPI were able to normalize EtOH-induced up-regulation of Nox and RANKL. In vitro experiments demonstrated that EtOH directly up-regulated Nox expression in osteoblasts. Pretreatment of osteoblasts with DPI eliminated EtOH-induced RANKL promoter activity. Furthermore, EtOH induced RANKL gene expression, and RANKL promoter activation in osteoblasts was ROS-dependent. These data suggest that inhibition of Nox expression and activity may be critical for prevention of chronic EtOH-induced osteoblast-dependent bone loss.
Footnotes
This work was supported in part by the National Institutes of Health National Institute of Alcohol and Alcoholism [Grant R01-AA18282] (to M.J.R.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.175091.
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ABBREVIATIONS:
- EtOH
- ethanol
- RANKL
- receptor activator of nuclear factor-κB ligand
- TNFα
- tumor necrosis factor α
- ROS
- reactive oxygen species
- Nox
- NADPH oxidase
- NAC
- N-acetyl cysteine
- DPI
- diphenylene iodonium
- TEN
- total enteral nutrition
- E2
- 17-β-estradiol
- pQCT
- peripheral quantitative computerized tomography
- BMD
- bone mineral density
- PCR
- polymerase chain reaction
- GAPDH
- glyceraldehyde 3-phosphate dehydrogenase
- ANOVA
- analysis of variance
- ER
- estrogen receptor
- ICI 182,780
- 7α-[9-[(4,4,5,5,5,-pentafluoropentyl)-sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17β-diol.
- Received September 15, 2010.
- Accepted November 18, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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