Peroxisome proliferator-activated receptor-alpha agonists repress expression of thrombin-activatable fibrinolysis inhibitor by decreasing transcript stability

2012 ◽  
Vol 108 (07) ◽  
pp. 74-85 ◽  
Author(s):  
Yutaka Masuda ◽  
Dan Saotome ◽  
Kimihiko Takada ◽  
Katsuyoshi Sugimoto ◽  
Tomoyuki Sasaki ◽  
...  
Keyword(s):  
Half Life ◽  
Hepg2 Cells ◽  
Gene Promoter ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Peroxisome Proliferator Activated Receptor ◽  
Fibrinolysis Inhibitor

SummaryThrombin-activatable fibrinolysis inhibitor (TAFI) (carboxypeptidase B2) is a plasma zymogen that is biosynthesised in the liver and released into the circulation. Activated TAFI is a prothrombotic factor which inhibits fibrin clot lysis. Cultured human hepatoma HepG2 cells were treated with peroxisome proliferator-activated receptor (PPAR)α, β or γ agonists, and the levels of TAFI antigen and mRNA (here, termed CPB2 mRNA) were measured. HepG2 cells treated with the PPARα agonist WY14643, but not agonists for PPARβ or PPARγ, decreased their release of TAFI antigen into the conditioned medium. In parallel, there were decreased levels of CPB2 mRNA and TAFI antigen in the cells. The WY14643-mediated decrease in CPB2 mRNA levels was accelerated by overexpression of PPARα and abolished by RNA interference of PPARA mRNA. CPB2 gene promoter activity was not influenced by treatment of the cells with WY14643. The half-life of the CPB2 transcript was shortened by treatment with WY14643 as compared with that of the control, and the decreased half-life of mRNA returned to control levels by treatment with a PPARα antagonist MK886 or transfection of PPARΑ-specific siRNA to WY14643-treated HepG2 cells. The present results suggest that PPARα agonists not only play a hypolipidaemic role, but also decrease the expression of TAFI, a prothrombotic factor, by decreasing stability of CPB2 transcripts.

Expression of thrombin-activatable fibrinolysis inhibitor (TAFI) is up-regulated by increase in intracellular cyclic AMP levels in cultured HepG2 cells

2009 ◽  
Vol 102 (12) ◽  
pp. 1204-1211 ◽  
Author(s):  
Yuka Kojima ◽  
Yutaka Masuda ◽  
Kimihiko Takada ◽  
Katsuyoshi Sugimoto ◽  
Tomohiro Ikeda ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Half Life ◽  
Hepg2 Cells ◽  
Promoter Activity ◽  
Intracellular Camp ◽  
Mrna Levels ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Carboxypeptidase B ◽  
Fibrinolysis Inhibitor ◽  
Human Hepatoma Hepg2 Cells

SummaryThrombin-activatable fibrinolysis inhibitor (TAFI), a carboxypeptidase B-like proenzyme, is predominantly biosynthesised in the liver and released into circulating plasma. Activated TAFI has a role in maintaining the balance between blood coagulation and fibrinolysis. We investigated the regulation of TAFI expression in cultured human hepatoma HepG2 cells. Stimulation of the cells with forskolin and dibutyryl cyclic AMP (DBcAMP) increased TAFI antigen levels in the cells in parallel with TAFI mRNA levels and antigen release from the cells into the conditioned medium. The elevated TAFI expression was abolished by pretreatment of the cells with KT5720, a protein kinase A (PKA) inhibitor. The promoter activity of the TAFI gene and the half-life of the TAFI transcript in DBcAMP-stimulated HepG2 cells increased to 1.5-fold and 2.0-fold, respectively, of those in the control cells. The increased promoter activity and the prolonged half-life were abolished by pretreatment of the cells with KT5720.These results suggest that an increase in intracellular cAMP levels upregulates TAFI expression in the cells in accompaniment with an elevation of TAFI mRNA levels, and that the elevated mRNA levels are derived from both transcriptional and post-transcriptional regulations of the TAFI gene mediated by activation of the AMP/PKA signaling pathway.

scholarly journals Evidence for differential effects of glucose and cycloheximide on mRNA levels of peroxisome proliferator-activated receptor- (PPAR-) machinery members: Superinduction of PPAR-gamma1 and -gamma2 mRNAs.

2010 ◽  
Vol 57 (2) ◽  
Author(s):  
Miroslav Rypka ◽  
Jaroslav Veselý
Keyword(s):  
Protective Effect ◽  
Hepg2 Cells ◽  
Actinomycin D ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Ppar Alpha ◽  
Peroxisome Proliferator Activated Receptor ◽  
Preferential Expression ◽  
Mrna Variant ◽  
Translational Inhibitor

Quantitative real-time RT-PCR study was conducted to reveal the effects of normal (5 mmol/l) and high (30 mmol/l) glucose without or with oleate (0.3 mmol/l) on mRNA levels of peroxisome proliferator-activated receptor- (PPAR-)alpha, -gamma1, -gamma2, and peroxisome proliferator-activated receptor-gamma coactivator- (PGC-)1alpha and -1beta in commercial human hepatoma-derived HepG2 cells maintained under low-serum condition. Significant decrease in PPAR-gamma1 and PGC-1alpha mRNA levels to about 50 % was observed during the first 4 h incubation period. During the next 4 h period, both PPAR-gamma1 and PGC-1alpha mRNAs were partly but significantly restored in high glucose batches. In this period, the presence of the transcriptional inhibitor actinomycin D revealed a significant protective effect of excess glucose on mature PPAR-gamma1 and PGC-1alpha mRNAs. Furthermore, PPAR-gamma1 and -gamma2 mRNAs were differentially superinduced 1.2-2.5 fold in cells upon the administration of the translational inhibitor cycloheximide. When the cells were co-treated with the combination of cycloheximide and actinomycin D, superinduction was completely suppressed, however. Altogether, the experiments revealed, first, an unexpected protective effect of abundant glucose on PPAR-gamma1 and PGC-1alpha mRNAs in HepG2 cells. Second, we demonstrated cycloheximide-induced, transcription-dependent upregulation of mature PPAR-gamma1 and -gamma2 mRNAs in HepG2 cells associated with preferential expression of the PPAR-gamma2 mRNA variant. The results draw attention to as yet unexplored mechanisms involved in the control of PPAR and PGC genes.

scholarly journals TGFβ1 Controls PPARγExpression, Transcriptional Potential, and Activity, in Part, through Smad3 Signaling in Murine Lung Fibroblasts

PPAR Research ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Allan Ramirez ◽  
Erin N. Ballard ◽  
Jesse Roman
Keyword(s):  
Transforming Growth Factor ◽  
Gene Promoter ◽  
Negative Regulator ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Reporter Expression ◽  
Peroxisome Proliferator Activated Receptor ◽  
3T3 Fibroblasts ◽  
Nih 3T3

Transforming growth factorβ1 (TGFβ1) promotes fibrosis by, among other mechanisms, activating quiescent fibroblasts into myofibroblasts and increasing the expression of extracellular matrices. Recent work suggests that peroxisome proliferator-activated receptorγ(PPARγ) is a negative regulator of TGFβ1-induced fibrotic events. We, however, hypothesized that antifibrotic pathways mediated by PPARγare influenced by TGFβ1, causing an imbalance towards fibrogenesis. Consistent with this, primary murine primary lung fibroblasts responded to TGFβ1 with a sustained downregulation of PPARγtranscripts. This effect was dampened in lung fibroblasts deficient in Smad3, a transcription factor that mediates many of the effects of TGFβ1. Paradoxically, TGFβ1 stimulated the activation of the PPARγgene promoter and induced the phosphorylation of PPARγin primary lung fibroblasts. The ability of TGFβ1 to modulate the transcriptional activity of PPARγwas then tested in NIH/3T3 fibroblasts containing a PPARγ-responsive luciferase reporter. In these cells, stimulation of TGFβ1 signals with a constitutively active TGFβ1 receptor transgene blunted PPARγ-dependent reporter expression induced by troglitazone, a PPARγactivator. Overexpression of PPARγprevented TGFβ1 repression of troglitazone-induced PPARγ-dependent gene transcription, whereas coexpression of PPARγand Smad3 transgenes recapitulated the TGFβ1 effects. We conclude that modulation of PPARγis controlled by TGFβ1, in part through Smad3 signals, involving regulation of PPARγexpression and transcriptional potential.

Plasma fibrin clot properties in the G20210A prothrombin mutation carriers following venous thromboembolism: the effect of rivaroxaban

2017 ◽  
Vol 117 (09) ◽  
pp. 1739-1749 ◽  
Author(s):  
Agnieszka Janion-Sadowska ◽  
Joanna Natorska ◽  
Jakub Siudut ◽  
Michal Zabczyk ◽  
Andrzej Stanisz ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Mutation Carriers ◽  
Lysis Time ◽  
Prothrombin Mutation ◽  
Fibrinolysis Inhibitor ◽  
Heterozygous Carriers ◽  
Mutation Group ◽  
Clot Structure

SummaryWe sought to investigate whether the G20210A prothrombin mutation modifies plasma fibrin clot properties in patients after venous thromboembolism (VTE) and how rivaroxaban treatment affects these alterations. We studied 34 prothrombin mutation heterozygous carriers and sex- and age-matched 34 non-carriers, all at least three months since the first VTE episode, before and during treatment with rivaroxaban. Clot permeability (Ks) and clot lysis time (CLT) with or without elimination of thrombin activatable fibrinolysis inhibitor (TAFI) were assessed at baseline, 2–6 hours (h) after and 20–25 h after intake of rivaroxaban (20 mg/day). At baseline, the prothrombin mutation group formed denser clots (Ks −12 %, p=0.0006) and had impaired fibrinolysis (CLT +14 %, p=0.004, and CLT-TAFI +13 %, p=0.03) compared with the no mutation group and were similar to those observed in 15 healthy unrelated prothrombin mutation carriers. The G20210A prothrombin mutation was the independent predictor for Ks and CLT before rivaroxaban intake. At 2–6 h after rivaroxaban intake, clot properties improved in both G20210A carriers and non-carriers (Ks +38 %, and +37 %, CLT −25 % and −25 %, CLT-TAFI −20 % and −24 %, respectively, all p<0.001), but those parameters were worse in the prothrombin mutation group (Ks −12.8 %, CLT +17 %, CLT-TAFI +13 %, all p<0.001). Rivaroxaban concentration correlated with fibrin clot properties. After 20–25 h since rivaroxaban intake most clot properties returned to baseline. Rivaroxaban-related differences in clot structure were confirmed by scanning electron microscopy images. In conclusion, rivaroxaban treatment, though improves fibrin clot properties, cannot abolish more prothrombotic fibrin clot phenotype observed in prothrombin mutation carriers following VTE.

Thyroid hormone is required for the phenotype transitions induced by the pharmacological inhibition of calcineurin in adult soleus muscle of rats

2008 ◽  
Vol 294 (1) ◽  
pp. E69-E77 ◽  
Author(s):  
Nathalie Koulmann ◽  
Lahoucine Bahi ◽  
Florence Ribera ◽  
Hervé Sanchez ◽  
Bernard Serrurier ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Soleus Muscle ◽  
Hormone Deficiency ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Peroxisome Proliferator Activated Receptor ◽  
Thyroid State ◽  
Novel Approach ◽  
A Subunit ◽  
Calcineurin Inhibition

The present experiment was designed to examine the effects of hypothyroidism and calcineurin inhibition induced by cyclosporin A (CsA) administration on both contractile and metabolic soleus muscle phenotypes, with a novel approach to the signaling pathway controlling mitochondrial biogenesis. Twenty-eight rats were randomly assigned to four groups, normothyroid, hypothyroid, and orally treated with either CsA (25 mg/kg, N-CsA and H-CsA) or vehicle (N-Vh and H-Vh), for 3 wk. Muscle phenotype was estimated by the MHC profile and activities of oxidative and glycolytic enzymes. We measured mRNA levels of the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), the major regulator of mitochondrial content. We also studied the expression of the catalytic A-subunit of calcineurin (CnA) both at protein and transcript levels and mRNA levels of modulatory calcineurin inhibitor proteins (MCIP)-1 and -2, which are differentially regulated by calcineurin activity and thyroid hormone, respectively. CsA-administration induced a slow-to-fast MHC transition limited to the type IIA isoform, which is associated with increased oxidative capacities. Hypothyroidism strongly decreased both the expression of fast MHC isoforms and oxidative capacities. Effects of CsA administration on muscle phenotype were blocked in conditions of thyroid hormone deficiency. Changes in the oxidative profile were strongly related to PGC-1α changes and associated with phosphorylation of p38 MAPK. Calcineurin and MCIPs mRNA levels were decreased by both hypothyroidism and CsA without additive effects. Taken together, these results suggest that adult muscle phenotype is primarily under the control of thyroid state. Physiological levels of thyroid hormone are required for the effects of calcineurin inhibition on slow oxidative muscle phenotype.

Effect of Heparin on TAFI-Dependent Inhibition of Fibrinolysis: Relative Importance of TAFIa Generated by Clot-Bound and Fluid Phase Thrombin

2002 ◽  
Vol 88 (08) ◽  
pp. 282-287 ◽  
Author(s):  
Anna Pentimone ◽  
Bianca Binetti ◽  
Marialisa Cramarossa ◽  
Donatella Piro ◽  
Nicola Semeraro ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Blood Clot ◽  
Fluid Phase ◽  
Clot Lysis ◽  
Relative Importance ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Dependent Inhibition ◽  
Fibrinolysis Inhibitor ◽  
Dependent Mechanism

SummaryHeparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 µg/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through “localized” TAFIa generation.

scholarly journals Liver nuclear hormone receptor PPAR, LXR and RXR expression and blood lipid and glucose levels in susceptible and resistent to hepatocarcinogenesis strain of mice

2010 ◽  
Vol 56 (4) ◽  
pp. 480-489
Author(s):  
E.N. Pivovarova ◽  
N.V. Baginskaya ◽  
M.L. Perepechaeva ◽  
S.I. Ilnitskaya ◽  
M.I. Dushkin
Keyword(s):  
Tumor Development ◽  
Corticosterone Level ◽  
Blood Lipid ◽  
Nuclear Hormone Receptor ◽  
Mouse Strains ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Peroxisome Proliferator Activated Receptor ◽  
Mrna Content ◽  
Dd Mice

Earlier it was shown that male mice of the DD/He strain were highly susceptible to ortho-aminoasotoluene (OAT) induced hepatocarcinogenesis, and resistant to spontaneous liver tumor development as compared to the СС57BR/Mv strain. In the present work we have made a comparative investigation of peroxisome proliferator-activated receptor (PPAR), liver X-receptor (LXR) and retinoic X-receptor (RXR) mRNA levels in liver as well as concentrations of corticosterone, glucose, lipids and insulin in blood of male DD/He and СС57BR/Mv mice. Using the multiplex RT-PCR method it was found that PPAR-α, PPAR-γ, RXR-α and RXR-β mRNA content was essentially decreased in the liver of DD mice as compared to mice of the СС57BR strain. No significant interstrain differences of LXR-α and LXR-β mRNA content were found. In DD micetere was more then the 3-fold decrease of blood content of corticosterone, which is involved in PPAR and RXR regulation. DD mice demonstrated a significant decrease in blood serum glucose and insulin concentrations as well as higher reactivity to insulin as compared with СС57BR mice. Elevated blood total cholesterol and cholesterol HDL level were found in DD mice whereas triglyceride content was basically the same in both mouse strains. It is known that glucocorticoids, PPAR and RXR play crucial role in transcription regulation of inflammation response. Therefore our data allow to suggest that decreased corticosterone level in blood, PPAR and RXR mRNA content in liver of the DD strain may lead to induction of inflammation by OAT exposure, resulting in a high incidence of tumorigenesis in this strain.

scholarly journals Molecular analysis of the human thrombin-activatable fibrinolysis inhibitor gene promoter

2007 ◽  
Vol 138 (2) ◽  
pp. 231-244 ◽  
Author(s):  
Mathieu Garand ◽  
Nazareth Bastajian ◽  
Michael E. Nesheim ◽  
Michael B. Boffa ◽  
Marlys L. Koschinsky
Keyword(s):  
Molecular Analysis ◽  
Gene Promoter ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Human Thrombin ◽  
Fibrinolysis Inhibitor ◽  
Inhibitor Gene

Identification and characterisation of monoclonal antibodies that impair the activation of human thrombin activatable fibrinolysis inhibitor through different mechanisms

2011 ◽  
Vol 106 (07) ◽  
pp. 90-101 ◽  
Author(s):  
Niraj Mishra ◽  
Ellen Vercauteren ◽  
Jan Develter ◽  
Riet Bammens ◽  
Paul J. Declerck ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Lysis Time ◽  
Human Thrombin ◽  
Fibrinolysis Inhibitor ◽  
Dose Dependent ◽  
Clot Lysis Time

SummaryThrombin activatable fibrinolysis inhibitor (TAFI) forms a molecular link between coagulation and fibrinolysis and is a putative target to develop profibrinolytic drugs. Out of a panel of monoclonal antibodies (MA) raised against TAFI-ACIIYQ, we selected MA-TCK11A9, MA-TCK22G2 and MA-TCK27A4, which revealed high affinity towards human TAFITI- wt. MA-TCK11A9 was able to inhibit mainly plasmin-mediated TAFI activation, MA-TCK22G2 inhibited plasmin- and thrombin-mediated TAFI activation and MA-TCK27A4 inhibited TAFI activation by plasmin, thrombin and thrombin/thrombomodulin (T/TM) in a dose-dependent manner. These MA did not interfere with TAFIa activity. Using an eightfold molar excess of MA over TAFI, all three MA were able to reduce clot lysis time significantly, i.e. in the presence of exogenous TM, MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 47 ± 9.1%, 80 ± 8.6% and 92 ± 14%, respectively, compared to PTCI. This effect was even more pronounced in the absence of TM i.e. MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 90 ± 14%, 140 ± 12% and 147 ± 29%, respectively, compared to PTCI. Mutagenesis analysis revealed that residues at position 268, 272 and 276 are involved in the binding of MA-TCK11A9, residues 147 and 148 in the binding of MA-TCK22G2 and residue 113 in the binding of MATCK27A4. The present study identified three MA, with distinct epitopes, that impair the activation of human TAFI and demonstrated that MATCK11A9 which mainly impairs plasmin-mediated TAFI activation can also reduce significantly clot lysis time in vitro.

Sign in / Sign up

Export Citation Format

Share Document