Novel mechanisms for the post-transcriptional regulation of monocyte-macrophage tissue factor expression

• Main Author: Iqbal, Bilal
• Other Authors: Haskard, Dorian
• Published: Imperial College London 2013
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.686265

Post-transcriptional regulation of mRNA is an important determinant of net gene expression. Tissue factor (TF) is the key stimulus for thrombus formation and plays critical roles in atherothrombosis. Little is known about its post-transcriptional regulation. Tristetraprolin (TTP) is the most widely studied mRNA-binding protein, and binds to the adenylate-uridylate rich elements (AREs) in the 3' untranslated region (3'UTR) of target mRNAs and promotes degradation. Poly(ADP-ribose)-polymerase-14 (PARP-14), belongs to a family of ~17 proteins with a PARP domain that generates negatively charged poly(ADP-ribose) adducts on intracellular proteins - a post-translational modification implicated in diverse cellular functions. Preliminary findings in our laboratory have identified PARP-14 as a novel RNA-binding protein, and found it to regulate inflammatory mRNA. This study sought to establish the roles for TTP and PARP-14 in regulating TF mRNA stability in LPS-stimulated monocyte-macrophages. Preliminary experiments demonstrated that TF mRNA, TF protein and TF mRNA stability were increased in Ttp-/- and Parp14-/- macrophages. Similarly, TF expression was increased in vivo in Parp14-/- mice. Furthermore, intravital microscopy demonstrated accelerated thrombosis following ferric-chloride injury in LPS-stimulated Parp14-/- mice. RNP immunoprecipitation, RNA biotin pulldown and co-immunoprecipitation assays demonstrated an interdependency for PARP-14 and TTP to form a ternary complex with TF mRNA. Both proteins interacted within the same 3'UTR segment containing a highly conserved palindromic ARE (AUAAUUUAUUUAAUA) comprising 2 overlapping AUUUAUUUA and UUAUUUAAU nonamers, both of which appeared to be critical in mediating this interaction. Inhibition of p38, which classically results in accelerated decay of TTP-regulated transcripts, reduced TF mRNA stability in WT but not Ttp-/- or Parp14-/- macrophages. Inhibition of PARP activity reduced TF mRNA stability in WT but not in Parp14-/- macrophages. Interestingly, PARP-14 conferred selectivity for TTP to degrade TF mRNA, as PARP-14 had no effect on TNFα expression, an established target of TTP. Similarly, PARP inhibition had no effect on TNFα mRNA decay. These data define novel roles for TTP and PARP-14 in the selective regulation of TF mRNA turnover, and demonstrates for the first time how the actions of TTP may be selectively regulated. PARP-14 functions as an accessory RNA-binding protein which is required for TTP to bind and degrade TF mRNA. Inhibition of either p38 or PARP catalytic activity accelerated TF mRNA decay. Thus one may propose that both PARP-14-mediated ADP-ribosylation and p38-mediated phosphorylation events are necessary for inactivating TTP-mediated TF mRNA decay. The selective destabilization of TF mRNA via pharmacological inhibition of PARP-14 may offer a novel therapeutic strategy in atherosclerosis and cardiovascular disease where thrombosis is linked to TF expression.