Modulation of CD40L expression by polypyrimidine tract binding protein (PTB) (51.8)

Abstract

The expression of CD40L is tightly regulated at the posttranscriptional level throughout a time course of T cell activation, which corresponds to a significant increase in message stability at late times of activation (24-48 hr). Previous work revealed a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex binds to the CD40L 3’UTR at late times of T cell activation. We used RNA interference to downregulate PTB in D1.1 Jurkat T cell line to address the functional roles of PTB in CD154 mRNA stability and surface expression. Real-time qPCR measurement of the CD154 mRNA decay revealed that downregulating PTB caused a > 2-fold decrease in the CD40L mRNA half-life. The downregulation of PTB also caused an approximate 2-fold decrease in the mean fluorescence (MFI) of CD40L. Analysis of PTB cellular distribution during a time course of CD4+ T cell activation revealed cytoplasmic and nuclear localization in all resting and activated cells. However, there was an increase in expression at late times of activation. Binding studies revealed that CD40L mRNA is bound by nuclear PTB at all times of activation indicating that the requirements for binding of CD40L message by nuclear versus cytoplasmic PTB is highly distinct. Finally, the binding of CD40L message corresponded to a change in phosphorylation status of cytoplasmic PTB. These findings support a model whereby activation-induced phosphorylation of PTB is required for cytoplasmic binding to CD40L mRNA.