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ARS Home » Southeast Area » Little Rock, Arkansas » Microbiome and Metabolism Research Unit » Research » Publications at this Location » Publication #208288
Title: Inhibition of reactive oxygen species generation and downstream activation of the ERK/STAT3/RANKL signaling cascade in osteoblasts accounts for the protective effects of estradiol on ethanol-induced bone loss

Author
item CHEN, JINRAN - ACNC/UAMS
item SHANKAR, KARTIK - ACNC/UAMS
item LIU, XIAOLI - ACNC/UAMS
item NAGARAJAN, SHANMUGAM - ACNC/UAMS
item CHEN, YING - ACNC/UAMS
item LUMPKIN, CHARLES - UAMS
item BADGER, THOMAS - ACNC/UAMS
item RONIS, MARTIN - ACNC/UAMS

Submitted to: Endocrine Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 2/15/2007
Publication Date: 6/2/2007
Citation: Chen, J., Shankar, K., Liu, X., Nagarajan, S., Chen, Y., Lumpkin, C.K., Badger, T.M., Ronis, M.J. 2007. Inhibition of reactive oxygen species generation and downstream activation of the ERK/STAT3/RANKL signaling cascade in osteoblasts accounts for the protective effects of estradiol on ethanol-induced bone loss [abstract]. The Endocrine Society, 89th Annual Meeting, June 2-5, 2007, Toronto, Canada. 2007 CDROM, Program No. P4-85.

Interpretive Summary: Alcohol is a significant component of the total caloric intake of a significant percentage of Americans. The vast majority of alcohol drinkers consume alcohol in moderate amounts, while a minority of people consumes alcohol to excess. Alcohol has biphasic health effects, with low daily intake having several documented health benefits and higher intake having adverse effects. Since alcohol accounts for a significant percentage of total calories and has significant biological affects that alter health outcomes, we are interested in defining the effects of low and high does alcohol on bone health. Our first studies have focused on high alcohol intake. It is well known that chronic and excessive alcohol intake will cause bone loss in females and males. We have previously found that the sex hormone estrogen can prevent bone loss due to chronic alcohol intake. The mechanism by which chronic alcohol consumption can cause bone loss and how estrogen can prevent chronic induced bone loss are unknown. In this study, we have conducted lab experiments that have answered the questions we raised above. We found that alcohol given to bone-forming cells could promote alcohol byproducts to accumulate into cells and cause certain genes to be "turned on" and cause more bone resorbing cells to form, and this cause bone breakdown. All these effects of alcohol on bone were blocked by treatment of cells with estrogen. We can conclude from these studies that estrogen can control genes that are turned-on in alcohol-treated bone-forming cells, thus providing explanation for results of previous studies and providing evidence for clinical treatment of alcohol-induced bone loss. Future studies will focus on other effects of alcohol and diet on bone health.

Technical Abstract: Bone loss occurs with chronic ethanol (EtOH) consumption in males and cycling females as a result of increased bone resorption. We have demonstrated that in vivo estradiol treatment can reverse this effect. However, the molecular mechanisms of EtOH-induced bone loss and of estrogen protection are largely unknown. We have investigated these mechanisms using primary stromal and calvaria osteoblasts from female neonatal rats and the rat osteoblast cell line UMR-106. Using osteoclast precursors from bone marrow and osteoblast/pre-osteoclast co-culture, we found that EtOH-induced RANKL in osteoblasts was able to promote osteoclastogenesis. The induction of RANKL by EtOH in stromal osteoblasts and stimulation of osteoclastogenesis could be blocked by pre-treatment of cells with either 17B-estradiol (E2) or the antioxidant N-acetyl cysteine (NAC). We used real-time reverse transcription PCR, flow cytometry, and western blotting, and determined that EtOH treatment of stromal osteoblasts increased the intracellular level of reactive oxygen species (ROS). This was associated with induction of nicotinamide adenine dinucleotide phosphate oxidase (Nox), and a downstream signaling cascade involving sustained activation of extracellular signal-regulated kinase (ERK) and activation of signal transducers and activators of transcription 3 (STAT3) resulting in increased gene transcription of RANKL. Interestingly, using reverse transcription PCR, we found that NOX1, NOX2, and NOX4, but not NOX3 were expressed in stromal osteoblasts. In the presence of EtOH, sustained nuclear ERK translocation > 24 h was observed in calvaria osteoblasts and UMR-106 cells transfected with GFP-ERK2. This was abolished by pretreatment with either E2 or NAC. Chemical inhibition of NOX by diphenylene iodonium in stromal osteoblasts also reversed the ability of EtOH to phosphorylate ERK and induce RANKL mRNA expression. Down-regulation of EtOH-induced ROS generation in osteoblasts was also observed following treatment with E2 or NAC. Therefore, interference with ROS generation and cytoplasmic kinase activation provides a molecular explanation for the preventive effects of E2 on EtOH-induced bone loss and suggests that in vivo antioxidant treatment would have similar protective effects.