Inhibition of protein kinase Cdelta 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 PKCdelta is one of the key isoenzymes in the regulation of inflammatory processes. In the present study, we investigated the role of PKCdelta in the regulation of the expression of tristetraprolin in activated macrophages by using the PKCdelta inhibitor, rottlerin, and by downregulating PKCdelta expression by using PKCdelta siRNA. TTP protein and mRNA expression were measured by Western blotting and quantitative RT-PCR, respectively. TTP and TNFalpha 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-kappaB, AP-2, SP1 and EGR1. Downregulation of PKCdelta by siRNA decreased significantly TTP expression in activated macrophages. Rottlerin also decreased TTP expression in wild type cells but not in cells in which PKCdelta 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-kappaB, Sp1, AP-2 or EGR1 (which have been shown to be involved in TTP transcription). In addition, rottlerin reduced the decay of TNFalpha mRNA, which is an important target of TTP. The results suggest that PKCdelta up-regulates the expression of TTP by stabilizing its mRNA which may serve as a feed-back loop to regulate the inflammatory response.
Leppänen
Tiina
T
The Immunopharmacology Research Group, University of Tampere Medical School and Research Unit, Tampere University Hospital, Tampere, Finland.
Jalonen
Ulla
U
Korhonen
Riku
R
Tuominen
Raimo K
RK
Moilanen
Eeva
E
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects*; D006207:Half-Life; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D051816:Tristetraprolin / Q000235:genetics* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D000098:Acetophenones; D001578:Benzopyrans; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha; C085746:rottlerin; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate