Pro-inflammatory cytokines reduce human TAFI expression via tristetraprolin-mediated mRNA destabilisation and decreased binding of HuR

Summary

Thrombin activatable fibrinolysis inhibitor (TAFI) is the zymogen form of a basic carboxypeptidase (TAFIa) with both anti-fibrinolytic and anti-inflammatory properties. The role of TAFI in inflammatory disease is multifaceted and involves modulation both of specific inflammatory mediators as well as of the behaviour of inflammatory cells. Moreover, as suggested by in vitro studies, inflammatory mediators are capable of regulating the expression of CPB2, the gene encoding TAFI. In this study we addressed the hypothesis that decreased TAFI levels observed in inflammation are due to post-transcriptional mechanisms. Treatment of human HepG2 cells with pro-inflammatory cytokines TNFα, IL-6 in combination with IL-1β, or with bacterial lipopolysaccharide (LPS) decreased TAFI protein levels by approximately two-fold over 24 to 48 hours of treatment. Conversely, treatment of HepG2 cells with the anti-inflammatory cytokine IL-10 increased TAFI protein levels by two-fold at both time points. We found that the mechanistic basis for this modulation of TAFI levels involves binding of tristetraprolin (TTP) to the CPB2 3′-UTR, which mediates CPB2 mRNA destabilisation. In this report we also identified that HuR, another ARE-binding protein but one that stabilises transcripts, is capable of binding the CBP2 3’UTR. We found that pro-inflammatory mediators reduce the occupancy of HuR on the CPB2 3’-UTR and that the mutation of the TTP binding site in this context abolishes this effect, although TTP and HuR appear to contact discrete binding sites. Interestingly, all of the mediators tested appear to increase TAFI protein expression in THP-1 macrophages, likewise through effects on CPB2 mRNA stability.

Keywords

Fibrinolysis inhibitors - inflammatory mediators - gene regulation - liver

• References

• 1 Anderson P. Post-transcriptional control of cytokine production.. Nat Immunol 2009; 09: 353-359.
• 2 Carrick DM, Lai WS, Blackshear PJ. The tandem CCCH zinc finger protein tristetraprolin and its relevance to cytokine mRNA turnover and arthritis. Arthritis Res Ther 2004; 06: 248-264.
• 3 Srikantan S, Gorospe M. HuR function in disease. Front Biosci 2012; 17: 189-205.
• 4 Foley JH, Kim PY, Mutch NJ, Gils A. Insights into thrombin activatable fibrinolysis inhibitor function and regulation. J Thromb Haemost 2013; 11 (Suppl. 01) 306-315.
• 5 Leung LL, Nishimura T, Myles T. Regulation of tissue inflammation by thrombin-activatable carboxypeptidase B (or TAFI). Adv Exp Med Biol 2008; 632: 61-69.
• 6 Boffa MB, Reid TS, Joo E. et al. Characterisation of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B). Biochemistry 1999; 38: 6547-6558.
• 7 Van Thiel DH, George M, Fareed J. Low levels of thrombin activatable fibrinolysis inhibitor (TAFI) in patients with chronic liver disease. Thromb Haemost 2001; 85: 667-670.
• 8 Bajzar L, Manuel R, Nesheim ME. Purification and characterisation of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 1995; 270: 14477-14484.
• 9 Mao SS, Cooper CM, Wood T. et al. Characterisation of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans. J Biol Chem 1999; 274: 35046-35052.
• 10 Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem 1996; 271: 16603-16608.
• 11 Redlitz A, Tan AK, Eaton DL, Plow EF. Plasma carboxypeptidases as regulators of the plasminogen system. J Clin Invest 1995; 96: 2534-2538.
• 12 Campbell WD, Lazoura E, Okada N, Okada H. Inactivation of C3a and C5a octapeptides by carboxypeptidase R and carboxypeptidase N. Microbiol Immunol 2002; 06: 131-134.
• 13 Hendriks D, Wang W, van Sande M, Scharpé S. Human serum carboxypeptidase U: a new kininase?. Agents Actions Suppl 1992; 38: 407-413.
• 14 Myles T, Nishimura T, Yun TH. et al. Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation. J Biol Chem 2003; 278: 51059-51067.
• 15 Du XY, Zabel BA, Myles T. et al. Regulation of chemerin bioactivity by plasma carboxypeptidase N, carboxypeptidase B (activated thrombin-activable fibrinolysis inhibitor), and platelets. J Biol Chem 2009; 284: 751-758.
• 16 Boffa MB, Hamill JD, Maret D. et al. Acute phase mediators modulate thrombin-activable fibrinolysis inhibitor (TAFI) gene expression in HepG2 cells. J Biol Chem 2003; 278: 9250-9257.
• 17 Lin JH, Garand M, Zagorac B. et al. Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells. Thromb Haemost 2011; 105: 999-1009.
• 18 Maret D, Boffa MB, Brien DF. et al. Role of mRNA transcript stability in modulation of expression of the gene encoding thrombin activable fibrinolysis inhibitor. J Thromb Haemost 2004; 02: 1969-1979.
• 19 Garand M, Bastajian N, Nesheim ME. et al. Molecular analysis of the human thrombin-activatable fibrinolysis inhibitor gene promoter. Br J Haematol 2007; 138: 231-244.
• 20 Boffa MB, Maret D, Hamill JD. et al. Effect of single nucleotide polymorphisms on expression of the gene encoding thrombin-activatable fibrinolysis inhibitor: a functional analysis. Blood 2008; 111: 183-189.
• 21 Novakovic D, Kuo AC, Lin JH. et al. Identification of tristetraprolin as a factor that modulates the stability of the TAFI transcript through binding to the 3’-untranslated region. J Thromb Haemost 2012; 10: 887-894.
• 22 Garand M, Lin JH, Zagorac B. et al. Regulation of the gene encoding human thrombin-activatable fibrinolysis inhibitor by estrogen and progesterone. Blood Coagul Fibrinolysis 2013; 24: 393-404.
• 23 Peng SS, Chen CY, Shyu AB. Functional characterisation of a non-AUUUA AU-rich element from the c-jun proto-oncogene mRNA: evidence for a novel class of AU-rich elements. Mol Cell Biol 1996; 16: 1490-1499.
• 24 Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992; 89: 5547-5551.
• 25 Urlinger S, Baron U, Thellmann M. et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 2000; 97: 7963-7968.
• 26 Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 1980; 25 209 497-499.
• 27 Kim PY, Foley J, Hsu G. et al. An assay for measuring functional activated thrombin-activatable fibrinolysis inhibitor in plasma. Anal Biochem 2008; 372: 32-40.
• 28 Lin JH, Novakovic D, Rizzo CM. et al. The mRNA encoding TAFI is alternatively spliced in different cell types and produces intracellular forms of the protein lacking TAFIa activity. Thromb Haemost 2013; 109: 1033-1044.
• 29 Galgano A, Forrer M, Jaskiewicz L. et al. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system. PLoS One 2008; 03: e3164.
• 30 Slobodin B, Gerst JE. A novel mRNA affinity purification technique for the identification of interacting proteins and transcripts in ribonucleoprotein complexes. RNA 2010; 16: 2277-2290.
• 31 Gutiérrez-Ruiz MC, Quiroz SC, Souza V. et al. Cytokines, growth factors, and oxidative stress in HepG2 cells treated with ethanol, acetaldehyde, and LPS. Toxicology 1999; 134: 197-207.
• 32 Hitti E, Iakovleva T, Brook M. et al. Mitogen-activated protein kinase-activated protein kinase 2 regulates tumor necrosis factor mRNA stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/ uridine-rich element. Mol Cell Biol 2006; 26: 2399-2407.
• 33 Tiedje C, Ronkina N, Tehrani M. et al. The p38/MK2-driven exchange between tristetraprolin and HuR regulates AU–rich element–dependent translation. PLoS Genet 2012; 08: e1002977.
• 34 Frère C, Morange PE, Saut N. et al. Quantification of thrombin activatable fibrinolysis inhibitor (TAFI) gene polymorphism effects on plasma levels of TAFI measured with assays insensitive to isoform-dependent artefact. Thromb Hae-most 2005; 94: 373-379.
• 35 Frère C, Tregouet DA, Morange PE. et al. Fine mapping of quantitative trait nucleotides underlying thrombin-activatable fibrinolysis inhibitor antigen levels by transethnic study. Blood 2006; 108: 1562-1568.
• 36 Juhan-Vague I, Renucci JF, Grimaux M. et al. Thrombin-activatable fibrinolysis inhibitor antigen levels and cardiovascular risk factors. Arterioscler Thromb Vasc Biol 2000; 20: 2156-2161.
• 37 Boffa MB, Koschinsky ML. Curiouser and curiouser: Recent advances in measurement of thrombin-activatable fibrinolysis inhibitor (TAFI) and in understanding its molecular genetics, gene regulation, and biological roles. Clin Biochem 2007; 40: 431-442.
• 38 Masuda Y, Saotome D, Takada K. et al. Peroxisome proliferator-activated receptor-alpha agonists repress expression of thrombin activable fibrinolysis inhibitor by decreasing transcript stability. Thromb Haemost 2012; 108: 74-85.
• 39 Uhlar CM, Whitehead AS, Serum Amyloid A. the major vertebrate acute-phase reactant. Eur J Biochem 1999; 265: 501-523.
• 40 Garand M, Lin JHH, Hill CE. et al. Regulation of the mouse gene encoding TAFI by TNF :role of NF B binding site. Cytokine 2012; 57: 380-397.
• 41 Lebedeva S, Jens M, Theil K. et al. Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR. Mol Cell 2011; 43: 340-352.
• 42 López de Silanes I, Zhan M. et al. Identification of a target RNA motif for RNA-binding protein HuR. Proc Natl Acad Sci USA 2004; 101: 2987-2992.
• 43 Srikantan S, Tominaga K, Gorospe M. Functional interplay between RNA-binding protein HuR and microRNAs. Curr Protein Pept Sci 2012; 13: 372-379.
• 44 Al-Ahmadi W, Al-Ghamdi M, Al-Souhibani N, Khabar KS. miR-29a inhibition normalizes HuR over-expression and aberrant AU-rich mRNA stability in invasive cancer. J Pathol 2013; 230: 28-38.
• 45 Meisner NC, Hackermuller J, Uhl V. et al. mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure. Chembiochem 2004; 05: 1432-1447.
• 46 Cheung ST, So EY, Chang D. et al. Interleukin-10 inhibits lipopolysaccharide induced miR-155 precursor stability and maturation. PLoS One 2013; 08: e71336.
• 47 Galbiati V, Carne A, Mitjans M, Galli CL, Marinovich M, Corsini E. Isoeugenol destabilizes IL-8 mRNA expression in THP-1 cells through induction of the negative regulator of mRNA stability tristetraprolin. Arch Toxicol 2012; 86: 239-248.
• 48 Cheng HS, Sivachandran N, Lau A. et al. MicroRNA-146 represses endothelial activation by inhibiting pro-inflammatory pathways. EMBO Mol Med 2013; 05: 949-966.