Although the bone remodeling cycle has long been thought to be the primary target for the deleterious effects of alcohol on bone integrity, a comprehensive examination of the effects of alcohol on bone remodeling-related gene expression has not been previously performed. We report here on the results of such an investigation and discuss the potential effect on bone of our finding that binge alcohol exposure causes significant differential expression of selected osteoblast or osteoclast-expressed genes with documented roles in bone remodeling.
Bone formation has long been believed to be the primary target of alcohol’s deleterious effects on bone metabolism [21
]. The expression of alkaline phosphatase and osteocalcin, both well-validated markers for bone formation activity, were significantly decreased after acute binge alcohol treatment in this study, suggesting that bone formation activity is depressed after binge alcohol exposure. Our data support previously reported effects of alcohol on bone marrow osteocalcin mRNA levels [22
] but contrast with other studies demonstrating transiently increased osteocalcin levels after a single alcohol injection in female rats [10
] or chronic alcohol feeding in adolescent male animals [4
]. Although differences in animal age and sex may account for these differences, a more likely explanation is that the binge-patterned high BALs achieved in the current study are more damaging to bone than lower BALs and cause a significant depression of bone formation activity not observed in previous studies. A recent study demonstrating that intermittent high-dose alcohol exposure by vapor inhalation also has significant detrimental effects on osteoblast function backs this supposition [23
]. The acute or lower chronic alcohol doses given to rats in the studies mentioned above may indeed be leading to a positive effect on osteoblast function, as suggested by the osteocalcin data presented in these studies. The mechanism underlying this positive effect of alcohol on markers of osteoblast function is currently not known, but this observation is supported by human longitudinal studies on factors modulating bone mass, which also show a positive effect of low-dose alcohol consumption on age-related maintenance of bone mass [24
The bone morphogenetic (BMP) family of proteins have well-documented anabolic effects on bone [25
]. We observed a general decrease in BMP gene expression after chronic binge alcohol treatment, with levels of BMP2, -4, and -5 and the receptor for BMP2 (BMP2r) significantly decreased. Decreased BMP mRNA expression has not been previously associated with alcohol-induced bone loss; it suggests that the decreases in bone formation observed after alcohol exposure [10
] may be due in part to decreased BMP-mediated signaling in osteoblasts. This result, coupled with decreases in the expression of several bone collagen genes and other bone formation-related genes observed here, suggests that one way that alcohol may affect bone formation is to cause a general decrease in osteoblast mRNA synthesis or the stability of transcripts required for bone formation-related protein synthesis. This mechanism could account for the decreased bone formation activity observed after alcohol exposure [21
]. Interestingly, Pthr1 expression levels were decreased by more than 40% after both acute and chronic binge exposure, suggesting that parathyroid hormone (PTH) signaling in osteoblasts may also be compromised by alcohol exposure. We previously demonstrated that binge alcohol-induced bone loss in rats was mitigated by high-dose intermittent PTH receptor administration [15
]. Taken together, these data suggest that exogenous intermittent PTH administration may be required to stimulate PTH-mediated osteoblast activity in alcohol-exposed bone.
We recently discovered that the canonical Wnt signaling pathway, which plays a critical role in bone formation [12
], may be a bone-specific target of binge alcohol exposure in adult rats [13
]. Analysis of our data found that the steady-state level of sclerostin mRNA, a Wnt inhibitor produced only by osteocytes [26
], is significantly increased after acute binge treatment. This is an intriguing finding because it suggests that alcohol exposure could also be targeting the osteocyte, causing these cells to increase production of sclerostin and effectively turning off canonical Wnt signaling in bone. This would have detrimental consequences on maintenance of bone mass as a result of decreases in the maturation of osteoblast precursors and subsequent bone formation, controlled by canonical Wnt target gene activity. Sost has also been shown to antagonize some BMP-mediated bone anabolic activity, which could further depress bone formation [26
Although osteoblasts and bone formation are generally accepted as the primary targets of alcohol in bone tissue, targeting of osteoclast regulation and bone resorption as a mechanism underlying alcohol’s deleterious effects on bone is less accepted. Recently published reports support a role for increased bone resorption after both chronic and binge alcohol exposure. Zhang and coworkers [7
] demonstrated increased bone resorption in mice after chronic alcohol exposure and determined that OPG, a soluble decoy receptor for RANKL produced by osteoblasts that functions as a modulator of bone resorptive activity, could block increased bone resorption induced by chronic alcohol exposure. Our laboratory demonstrated increased bone resorption in young adult male rats by measuring serum deoxypyrodinoline levels after exposure to repeated binge alcohol cycles [8
]. Treatment with the antiresorptive bisphosphonate risedronate prevented not only binge alcohol-induced increases in serum deoxypyrodinoline, but also decreases in cancellous BMD and compressive strength associated with binge alcohol treatment. Chen and coworkers recently demonstrated increased bone resorption and RANKL mRNA expression after chronic alcohol feeding in female rats and in primary cultured bone cells [22
], suggesting that an increase in osteoblast RANKL expression may be the mechanism responsible for increased bone resorptive activity associated with alcohol.
We observed differential expression of two key regulators of osteoclast maturation, RANKL and OPG. The RANKL-OPG system is widely recognized as vital to osteoclast differentiation and resorption activity [28
]. This observation suggests a molecular mechanism whereby alcohol treatment could stimulate osteoclast differentiation and activation through increasing RANKL-mediated signaling and decrease the attenuating effect of OPG, leading to increased bone resorption. Because both RANKL and OPG are made by osteoblasts, these data support the concept that osteoblast-mediated bone formation and resorption regulatory activities may be the key targets of alcohol in bone. The expression of RANK, the receptor for RANKL found on osteoclast cells, shows decreased expression after a 4-week alcohol binge, possibly representing the activation of a negative feedback loop modulating an alcohol-related increase in osteoclast activity. Although increased RANKL expression observed in this study is in agreement with previous reports [22
], our observation that OPG mRNA levels are depressed by bingelike alcohol exposure is novel and supports the concept that intermittent binge alcohol exposure may be especially disruptive to osteoblast function. Recent evidence suggesting that OPG expression is regulated by Wnt/β
-catenin signaling [30
] suggests that the effect of alcohol on OPG expression that we demonstrate here is controlled through the alcohol-related targeting of the canonical Wnt pathway that we have previously demonstrated [13
IL-6 and OSM are inflammatory cytokines that stimulate bone resorptive activity [31
] by increasing expression of RANKL and decreasing OPG expression [32
]. Our results demonstrate significantly increased IL-6 and OSM expression levels after chronic binge alcohol, suggesting that binge alcohol stimulation of inflammatory cytokines could be responsible for the observed modulation of RANKL and OPG levels, although increased RANKL expression precedes the increases in IL-6 and OSM observed here.
The effect of chronic binge alcohol treatment on final posttreatment animal weight is a limitation to the current study that could affect the interpretation or relevance of our results. We addressed this concern, as described in Results, by statistical regression analysis, which revealed no significant contribution of weight to the observed decreases in vertebral BMD and strength, and which revealed highly significant treatment (i.e., alcohol)-related effects on bone integrity. This analysis strongly suggests that alcohol treatment has a damaging effect on bone in this experimental paradigm and that it is likely either directly or indirectly responsible for the changes in bone remodeling-related gene expression presented here. The fact that binge alcohol-exposed rats gained weight throughout the study period suggests that animals were in good overall health and were consuming enough food during the study period. Although not measured in this study, no significant differences in weekly food intake were observed between control and binge alcohol-treated adult animals in a previous investigation [15
]. A direct effect of alcohol exposure on weight gain was observed in a previous study by our laboratory in animals exposed to chronic alcohol administration using the Lieber-DeCarli liquid diet, with control animals given a liquid diet matched to the caloric intake of their alcohol-fed counterparts, eliminating the potential confounding of reduced caloric intake by alcohol-exposed animals [33
In summary, analysis of bone formation- and resorption-specific gene expression profiles after binge alcohol treatment demonstrates a general trend of decreased differential expression for several bone formation-related genes and the modulation of important regulators of bone resorption. This information adds to our understanding of the mechanisms underlying alcohol-induced bone loss by demonstrating that on a molecular level, alcohol targets the expression of genes with well-defined roles in bone remodeling. Therapeutic agents targeting bone remodeling have found clinical utility in the treatment of other metabolic bone diseases such as osteoporosis; this study demonstrates that current and future therapeutic agents targeting bone turnover should be useful in treating or preventing alcohol-related bone loss. This information may also be useful in designing the next generation of drugs that target the unique aspects of alcoholic bone disease.