This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. SMS represents an important class of enzymes responsible for the biosynthesis of sphingomyelin (SM), a critical structural component of the plasma membrane. Moreover, in the course of its biochemical reaction, SMS also regulates, in opposing directions, the levels of two important bioactive lipids, ceramide and diacylglycerol (DAG), which often play antagonistic roles in the control of key cellular functions such as proliferation and/or induction of cell death. Recently two enzymes, SMS1 and SMS2 have been proposed as potential SMSs based on biochemical characterization studies using a yeast expression system. Soon after, SMS1 was proved to mediate SM synthesis in mammalian cells. Previous studies conducted by the PI showed that activation of SM synthesis coupled with activation of NF-kB, a pivotal transcription factor, involved in the regulation of cellular functions, such as survival and inflammatory response. Our results obtained during year 1 of the present project characterized SMS2 as a SMS in mammalian cells, thus demonstrating that both SMS1 and SMS2 are capable of SMS activity in mammalian cells. Additionally, we found that knock-down of SMS2 significantly impaired basal and cytokine induced-activation of NF-kB in a PKC-independent manner, with consequent inhibition of the expression of NF-kB-responsive genes, such as the cyclooxigenase 2 gene (cox-2). Based on the potential role of SMS2 in promoting NF-kB activation and because NF-kB activation has been identified as a key factor promoting cancer development, we HYPOTHESIZE that SMS regulates activation of the prosurvival transcription factor NF-kB, through modulation of the bioactive lipids controlled by its biochemical reaction, and that SMS is therefore a determinant for the development of cancer. RESULTS: In vivo biochemical characterization of SMS2 established that, under basal conditions, the golgi sub pool of SMS2 is active whereas still no evidence is collected on the activity of the plasma membrane localized SMS2 pool. Both SMS1 and SMS2 regulate NF-kB activity in a similar fashion and independently from IkB degradation. In transformed phenotypes such as chronic myelogeneous leukemia and SV40-transformation, the expression levels of SMS were increased. In the CML model the increase in SMS1 expression was not due to increased message stability suggesting an enhanced transcription. INTERPRETATION AND DIRECTION: The fact that SMSs seem to regulate NF-kB in a non classical mode, by-passing regulation by IkB, shed light and underlie the importance of an additional and still ill-studied step in the regulation of NF-kB. Therefore the significance of our results is two-fold: they establish NF-kB as a target for SMS1 and SMS2 activity, and they are starting to elucidate an additional level of regulation of NF-kB activity down-stream of IkB. Moreover, the fact that in different transformed phenotypes SMSs expression is elevated, and that in bcr-abl cells, modulation of SMS1 expression results in modulation of cellular viability, underscores the fact that elevated SMS activity might represent a requisite to favor cell transformation or survival perhaps in harmful conditions.
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