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.
We have previously shown that the RNA binding protein, polypyrimidine tract-binding protein (PTB) plays a crucial role in regulating the expression of CD40L in activated CD4 T cells. Regulation occurs through stabilizing the message at late times of activation as well as changing the distribution of CD40L mRNA within distinct cellular compartments. Further experiments were designed to assess how PTB affects global CD4 T cell activation by using shRNA introduced into primary human CD4 T cells. Specifically, we found that PTB is critical for the proliferation and viability of activated T cells. The propensity to undergo cell death was evident in both infected and uninfected CD4 T cells within the same population suggesting that either soluble and/or cognate interactions were being affected by decreased PTB. Accordingly, PTB was found to be required for optimal expression of CD25, CD69, TNF-α and IL-2 but not CD38 or IFN-γ. When the decay rates were assessed in anti-CD3/anti-CD28-stimulated CD4 T cells, the pattern of IL-2, TNF-α and CD69 mRNA decay did not change over a time course of activation whereas CD25 showed a pattern of mRNA decay that was highly similar to CD40L. Evaluation of signaling pathways using phosphoflow analysis revealed PTB-specific changes in the NF-κB and MAPK-ERK pathways. Together, these findings support a role for PTB in the control of CD4 T cell activation through processes that are both dependent on and independent of PTB-regulated mRNA stability.
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