Inhibition of protein kinase Cδ reduces tristetraprolin expression by destabilizing its mRNA in activated macrophages

Tristetraprolin (TTP) binds to AU-rich elements within the mRNAs of several inflammatory genes and causes destabilization of the target mRNAs. The protein kinase C (PKC) pathway represents a major signalling system in inflammation and PKCδ is one of the key isoenzymes in the regulation of inflammatory processes. In the present study, we investigated the role of PKCδ in the regulation of the expression of tristetraprolin in activated macrophages by using the PKCδ inhibitor, rottlerin, and by downregulating PKCδ expression by using PKCδ siRNA. TTP protein and mRNA expression were measured by Western blotting and quantitative RT-PCR, respectively. TTP and TNFα mRNA decays were studied by the actinomycin D assay. In addition, we measured nuclear translocation of transcription factors believed to be important for TTP transcription, i.e. NF-κB, AP-2, SP1 and EGR1. Downregulation of PKCδ by siRNA decreased significantly TTP expression in activated macrophages. Rottlerin also decreased TTP expression in wild type cells but not in cells in which PKCδ had been downregulated by siRNA. Rottlerin decreased TTP mRNA half-life as measured by actinomycin D assay but it did not affect the nuclear translocation of transcription factors NF-κB, Sp1, AP-2 or EGR1 (which have been shown to be involved in TTP transcription). In addition, rottlerin reduced the decay of TNFα mRNA, which is an important target of TTP. The results suggest that PKCδ up-regulates the expression of TTP by stabilizing its mRNA which may serve as a feed-back loop to regulate the inflammatory response.