Predoctoral Fellowship Rutkute: Hepatocellular Mechanisms of Cytokine-Mediated CRP Production Upregulation During Aging

serum CRP levels, which lead to increased incidence of coronary heart disease and stroke in the elderly. We have recently found that hepatocy1es from aged animals exhibit dramatically upregulated response to IL-1beta, a major inducer of CRP production. Thus, it is possible that aging hypersensitizes cells to IL-1beta, thereby resulting in CRP production even in the absence of clinically relevant inflammation. The objective of this study is to understand the mechanism of the increased responsiveness to IL-1beta in aged hepatocy1es. Neutral sphingomyelinase (N-SMase), which positively modulates the transcription of IL-1beta target genes, exhibits significantly higher activity in livers of aged animals. This phenomenon may be due to depletion of intracellular glutathione (GSH), a potent inhibitor N-SMase, by age-related oxidative stress. Therefore, we hypothesize that age-related GSH depletion upregulates the activity of N-SMase, thereby inducing the hyperresponsiveness to IL-1beta, which may result in increased CRP production. Two specific aims are proposed: 1) Determine whether N-SMase is required and sufficient for the induction of the age-related hyperresponsiveness to IL-1beta; 2) Determine whether GSH depletion is responsible for the age-related upregulation of N-SMase activity. The studies will be performed in primary hepatocy1es isolated from young and aged rats. For Specific Aim 1, N-SMase will be overexpressed by adenovirus-mediated transfer in young rat hepatocy1es, and its expression will be silenced by morpholinos in aged rat hepatocy1es. It is expected that the resulting changes in N-SMase expression will positively or negatively, respectively, affect the activation of IL-1beta downstream targets such as JNK1/2 and transcription factors AP-1 and NF-kappaB, and it will subsequently modulate CRP mRNA levels. For Specific Aim 2, GSH will be depleted in young hepatocy1es, or supplemented in aged hepatocy1es. It is anticipated that changes in GSH contents will translate into N-SMase activity changes, and it will subsequently affect the activation of the IL-1beta targets and CRP mRNA levels. This study is expected to contribute to the understanding of dysregulated CRP production in the elderly, and it may place N-SMase among the therapeutic targets for the treatment of chronically elevated CRP and its associated cardiovascular conditions.