Rozenova Fellowship: The role of Acid Sphingomyelinase as a regulator of TNF-alpha levels in endotoxic shock model

Tumor necrosis factor alpha (TNF-a) is a major mediator of inflammation and plays an important role in the onset of many cardiovascular diseases. Its up-regulation during heart failure, atherosclerosis, etc. is linked to the onset and progression of these diseases, suggesting promising results from usage of TNF-a anatagonists as a therapeutic. Long term clinical trials, however did not show any benefits, emphasizing the dual, protective and damaging, effects of TNF-a, as well as the complex regulation of its production and clearance. Here we report that upon LPS administration, acid sphingomyelinase knockout (ASMase(-/-) ) mice exhibited 10-15 fold higher levels of TNF-a as compared to litter-matched controls. Furthermore, in in vitro studies ASMase deficiency correlates to increased synthesis and secretion of TNF-a. Importantly, restoration of ceramide in ASMase(-/-) cells by adding exogenous ceramide reduced the levels of secreted TNF-a in a dose dependent manner, suggesting a role for ASMase as a negative regulator of TNF-a production. Thus, the objective of this proposal is to decipher the mechanism by which ASMase regulates TNF-a levels in response to LPS stimulation. Two specific aims are proposed to address this question. The goal of specific aim 1 is to determine which level of TNF-a production is regulated by ASMase. Macrophage cell line RAW264.7 and peritoneal macrophages isolated from ASMase(-/-) and ASMase(+/+) mice will be used as in vitro model. The effect of ASMase deficiency and overexpression on activation of TNF-a promoter, mRNA stabilization and proteolitic cleavage of membrane associated TNF-a will be examined by luciferase assay, real time PCR and pulse chase labeling. In specific aim 2 we will test the hypothesis that ASMase deficiency also results in increased levels of secreted p75(sp75), therefore interfering with TNF-a inactivation and clearance. Our final conclusions will be based on results from in vivo studies including measurement of sp75 in serum from LPS injected ASMase(-/-) and ASMase(+/+) mice, as well as TNF-a cytotoxicity assay. This will be supported by in vitro study of ASMase effects on p75 expression and shedding. This study is expected to provide another insight on the regulation of TNF-a production and shed light on the physiological meaning of the correlation between the levels of sp75 and TNF-a activation state and clearance.