NF-kB dependent Fas signaling in hepatocytes

Quoc Tran, Rohini Mehta, A. Birerdinc. A. Baranova

This is a collaborative project between

Molecular and Microbiology Department, College of Science,George Mason University, Fairfax, VA

Translational Reseach Institute, Inova Hospital, VA

High prevalence and substantial morbidity and mortality accompanying NAFLD (Non alcoholic fatty liver disease) makes it imminent to understand the mechanistic basis of this disorder. Hepatocyte apoptosis, is an important mechanism in the pathogenesis of NAFLD with Fas signaling being an important player. The focus of the current study is to determine whether chemokines CCL21 and CCL4 induce Fas ligation and, if so, whether the Fas signaling activates downstream NF-kB mediated inflammatory pathway, or caspase-3 dependent apoptosis. HepG2 cell line (ATCC No. HB-8065 Hep G2 ;Hepatocellular carcinoma, human) will be used as in vitro model for the current study. The cells will be subcultured according to the recommended protocol (ATCC). Cells will be subjected to varying concentration of individual chemokines in a time course experiment of 8, 12 and 24 hours. After stimulation as described above, supernatants will be collected and subjected to ELISA using custom Multi-analyte ELISArray kits from Qiagen (Fredericks, MD) according to the manufacturer's suggestions. The cytokines represented by these arrays would include: IL1β, IL6, MIP-1α, MIP-1β, RANTES, TNFα which are target of transcriptional factor NF-kB. Experiments will be performed in triplicates. Delineating the previously unexplored role of CCL21 and CCL4 could help elucidate whether these chemokines act upstream of inflammation or are the targets of inflammatory pathways. It is plausible that Fas system via NF-kB may induce chemokine production triggering inflammation or aggravating existing inflammatory response. This could be a critical factor influencing the progression of NAFLD.