Molecular Mechanism Of TNF Alpha Effect On Organic Anion Transporting Polypeptide 1A2 Expression

Organic anion transporting polypeptides is are important membrane transporter proteins that mediate endogenous and exogenous substances across cell membrane. They are key determinants for absorption, distribution and excretion of numerous clinical drugs. As an importantmember of the OATP family, OATP1A2 expresses in different tissues such as the brain,intestine,liver, and kidney. It can transport wide spectrum of xenobiotics and thus affect their accumulation within cells. Therefore, regulation of OATP1A2 expression would influence bioavailability of various drugs. Studies have shown that inflammationmay down-regulate the expression and function of drug metabolizing enzymes and transporter, hence altering drug disposal process of the body. A series of inflammatory cytokines such as TNFa were suggested to be involved in such a process. However, the underlying molecular mechanism(s) are still unclear. In the current study, we analyzed around 2kb of the upstream sequence of OATP1A2 coding gene SLCO1A2 and found out that there is a consensus sequence that is corresponding to a putative NFκB binding site. Since regulation by TNFα is often mediated through the NFκB signal pathway, we further analyzed the role of this site in the process of TNFα regulation of OATP1A2.The results obtained are as follows:1) Constructs containing different length of upstream sequence of SLCO1A2 were generated and it was found that recombinant plasmid with the sequence that contained putative NFκB binding sites exhibited lower luciferase activity than those without such a binding site, suggesting that binding of NFκB with the position may have inhibitory effect on OATP1A2expression;2) Luciferase activity was partially recovered after critical bases of the putative NFκB binding site were mutated, which implicated that this site indeed plays a role in regulation of OATP1A2 expression;3) Luciferase activity was partially recovered after cells expressing luciferase reporter constructs that contains the putative NFκB binding site were treated with TNFα neutralizing antibody, further proved that this site is involved in regulation of OATP1A2 by TNFα;4) We proved the interaction of the putative binding site with NFκB through chromatin immunoprecipitation. It was also demonstrated that in the presence of TNFα, there was an increased amount of NFκB interacts with the position. These data indicated that TNFα may affect expression of OATP1A2 through a negative regulation by NFκB.