It is well established that chronic ethanol exposure can induce osteoporosis, a debilitating and costly disease. However, the molecular mechanisms through which ethanol achieves its influence on bone is unknown. There is evidence that ethanol may both increase bone resorption and inhibit bone production, resulting in osteopenia. The proinflammatory cytokine, interleukin-6 (IL-6), is an important mediator of osteoclast activity on bone. Furthermore, the investigators provide evidence that ethanol induces IL-6 expression and IL-6 promoter activity in a human bone marrow stromal cell line and increases serum IL-6 levels in mice. Accordingly, they hypothesize that ethanol induces IL-6 gene transcription in bone and the bone microenvironment culminating in osteoclast-mediated bone resorption. To explore this hypothesis, the investigators will perform the following specific aims: 1. Elucidate the mechanism of ethanol s influence on transcriptional activity of the IL-6 promoter in human bone marrow stromal cells; 2. Determine the degree and method of ethanol s influence on IL-6 mRNA stability in human bone marrow stromal cells; 3. Demonstrate that IL-6 contributes to the mechanism of ethanol-induced bone loss in vivo in mice.
Aim 1 will be accomplished by a) evaluation of ethanol s effect on nuclear transcript production, b) mutational analysis of the IL-6 promoter for ethanol-responsive cis-acting elements, and c) measurement of ethanol-mediated changes in transcription factor activity in a human marrow stromal cell line and primary human stromal cells.
Aim 2 will be accomplished by evaluation of a) ethanol induced modulation mRNA half-life, b) mutational analysis of the 3-untranslated region (UTR) of the IL-6 mRNA to identify regions effected by ethanol, and c) identification of ethanol-induced changes in trans-acting factors on the 3 -UTR in human marrow stromal cell line and primary human stromal cells.
Aim 3 will be accomplished by evaluating for differences in bone metabolism in mice which cannot express IL-6 (IL-6 gene knockout) and control mice (which can express IL-6) fed a liquid ethanol diet for 3 or 24 weeks. Bone metabolism will be evaluated by a variety of measurements including alterations in bone mineral content as assessed by total skeletal ash, bone marrow osteoclast production by marrow culture, osteoclast activity by pit resorption assay, in vivo bone resorption by urinary pyridinoline crosslinks, and osteoclast activity by bone histomorphometry.
