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Monocyte–derived tissue factor (TF) 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 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. We have recently identified PARP–14 as a novel RNA–binding protein.
We sought to determine the roles for TTP and PARP–14 in regulating TF expression, and the potential role of ADP ribosylation in TF mRNA turnover.
TF mRNA and protein expression were increased in TTP–/– vs TTP+/+ macrophages (p<0.05) with increased TF mRNA stability (t1/2=347±62min vs 82±14min, p<0.001). Similarly, TF mRNA and protein levels were increased in PARP14–/– vs. PARP–14+/+ macrophages (p<0.05) with increased TF mRNA stability (t1/2=181±11min vs 60±5min, p<0.001). TF mRNA, activity and protein were increased in vivo (heart, lung, kidney and aorta) in PARP–14–/– vs PARP–14+/+ mice (p<0.05). Intravital microscopy demonstrated a 66% reduction in median arteriolar occlusion time in LPS–stimulated PARP–14–/– vs PARP–14+/+ mice following ferric–chloride injury (p=0.008). RNP immunoprecipitation and RNA biotin pulldown assays demonstrated an interdependency for PARP–14 and TTP to form a ternary complex with TF mRNA. Inhibition of PARP activity reduced TF mRNA stability in PARP–14+/+ macrophages (p=0.018), but not in PARP–14–/– macrophages.
These data define novel roles for TTP and PARP–14 in regulating TF mRNA turnover. PARP–14 functions as an accessory RNA–binding protein which is required for TTP to bind and degrade TF mRNA. Furthermore, inhibition of PARP–14 catalytic activity promotes TTP–mediated TF mRNA degradation. Thus destabilization of TF mRNA via pharmacological inhibition of PARP–14 may offer a novel therapeutic strategy in atherothrombosis where thrombosis is linked to TF expression.
Special Session North, Room 120
Sunday, March 10, 2013, 8:00 a.m.–8:15 a.m.
Session Title: Young Investigator Awards Competition: ACCF/Herman K. Gold Young Investigators Award in Molecular and Cellular Cardiology
Abstract Category: Molecular and Cellular Cardiology
Presentation Number: 402–4
- 2013 American College of Cardiology Foundation