The habitual consumption of even moderate quantities of alcoholic beverages (one to two drinks per day) can result in a host of abnormal clinical, biochemical, and physiologic findings that stem from the toxic effects of alcohol on the liver, marrow, brain and skeleton among others. A pathological effect of ethanol on cellular proliferation has been described in all of these target tissues. Yet, the investigation of alcohol-induced growth suppression at the cellular level has, thus far, failed to uncover the mechanism of this adverse effect. The PI's laboratory has confirmed the inhibitory effect of clinically relevant concentrations of ethanol on cell proliferation in vitro in a well-defined osteoblast model system. Additional study of this effect revealed that the growth suppression induced by ethanol was paralleled by suppression of mitogen-activated protein (MAP) kinase activity. The MAP kinase protein phosphorylation cascade plays an essential role in triggering cell proliferation and activation of MAP kinase occurs through successive stimulation of specific proteins in the Ras activation pathway (Ras, Raf-1, and MAP kinase). However, ethanol exerts no inhibitory effect on MAP kinase activity in cells overexpressing a constitutively active Ras gene. These observations indicate that ethanol interferes with an important site in the proliferation pathway that is proximal to the action of Ras. Ras activation is necessary for mitogenesis induced by the receptor tyrosine kinases and, recently, the involvement of adapter molecules and GTP exchange proteins for Ras has been experimentally described. Based on these intriguing observations, it is hypothesized that ethanol inhibits cellular proliferation by interfering with a specific site in the tyrosine kinase phosphorylation pathway that leads to the activation of Ras. The specific objectives of this proposal are : (1) to characterize the intracellular signaling pathway necessary for growth factor-dependent activation of Ras in the osteoblast(s) that is perturbed by ethanol; (2) to identify the site(s) of in the growth factor receptor tyrosine kinase pathway affected by ethanol; (3) to determine the molecular mechanism(s) by which ethanol interferes with growth factor tyrosine kinase signaling pathway in the osteoblast. Ethanol inhibits growth in a wide variety of tissues and the IGF axis is ubiquitous, regulating growth and development of all cell types. Our observations in this model bone cell system are likely to provide fresh insights into the anti-proliferative actions of ethanol in general.
