Abstract 805: Differential Regulation Of Cytokine-induced Alternative Splicing Of The TF Gene By Serine/Arginine Rich Protein Kinases

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ursula Rauch ◽  
Andreas Eisenreich ◽  
Wolfgang Poller ◽  
Heinz-Peter Schultheiss
Keyword(s):  
Alternative Splicing ◽  
Protein Phosphorylation ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Splice Variant ◽  
Sr Protein ◽  
Phosphorylation State ◽  
Post Induction ◽  
Tnf Α ◽  
Tf Gene

Background: Higher eukaryotes control gene expression and increase protein diversity by alternative splicing of pre-mRNA. The Cdc2-like kinase (Clk) family, DNA topoisomerase I (DNA topo I) or Akt kinase are involved in splicing control by regulating the phosphorylation state of serine/arginine rich (SR) proteins. We recently showed that alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), the primary initiator of coagulation, is expressed in HUVECs in response to inflammatory cytokines. This study investigated the role of Clks, DNA topo I and the PI3K-Pathway in regulation of TF-splicing in TNF-α induced HUVECs. Methods: HUVECs were incubated with inhibitors of Clks, DNA-topo I or PI3K and were then stimulated with TNF-α. The SR protein phosphorylation state was determined 2 min post induction. The full length (fl) TF and asHTF mRNA were assessed 60 min post induction by Real-Time PCR. Proteins were measured 5 and 8 hours after stimulation by Western blots and the cell thrombogenicity was analyzed via a chromogenic assay. Results: TNF-α inceased the mRNA expression of asHTF and flTF in HUVECs. The Clk-inhibitor completely inhibited the TNF-α induced expression of asHTF and reduced flTF by 30 %. Inhibition of DNA topo I increased asHTF expression and reduced the flTF expression. Inhibition of the PI3K/Akt-pathway had no effect on TF mRNA expression. Reduced Clk-inhibition the TF activity by 50 % whereas DNA topo I inhibition significantly decreased the procoagulant TF activity 8 hours post TNF-α induction. The Clk- and DNA-topo I-inhibitors altered the SR-protein phosphorylation pattern post TNF-α-induction. Additionally resulted inhibition of Clks in the generation of a third TF mRNA-splice variant, TF-A. Conclusion: Selective inhibition of Clks or DNA topo I leads to alterations of SR-protein phosphorylation and affects the differential expression of TF isoforms, thereby modulating the thrombogenicity of HUVECs. The inhibition of Clks contributes to the generation of a third TF splice variant. The inhibition of these kinases gives new insights into the regulation of the TF gene splicing process, which may result in new therapeutic strategies for modulating cellular thrombogenicity.

Alternative Splicing Is Responsible for the Presence of Two Tissue Factor mRNA Species in LPS Stimulated Human Monocytes

1992 ◽  
Vol 67 (02) ◽  
pp. 272-276 ◽  
Author(s):  
C Paul ◽  
E van der Logt ◽  
Pieter H Reitsma ◽  
Rogier M Bertina
Keyword(s):  
Alternative Splicing ◽  
Tissue Factor ◽  
Stop Codon ◽  
Cell Types ◽  
Northern Analysis ◽  
Human Monocytes ◽  
Mrna Species ◽  
Protein Levels ◽  
Intron 1 ◽  
Tf Gene

SummaryAlthough normally absent from the surface of all circulating cell types, tissue factor (TF) can be induced to appear on circulating monocytes by stimulants like bacterial lipopolysaccharide (LPS) and phorbolesters. Northern analysis of RNA isolated from LPS stimulated human monocytes demonstrates the presence of 2.2 kb and 3.1 kb TF mRNA species. The 2.2 kb message codes for the TF protein. As demonstrated by Northern blot analysis with a variety of TF gene probes, the 3.1 kb message arises from an alternative splicing process which fails to remove 955 bp from intron 1. Because of a stop codon in intron 1 no TF protein is produced from the 3.1 kb transcript. This larger transcript should therefore not be taken into account when comparing TF gene transcription and TF protein levels.

scholarly journals Retinoic Acid Selectively Inhibits Lipopolysaccharide Induction of Tissue Factor Gene Expression in Human Monocytes

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2857-2865 ◽  
Author(s):  
Paul Oeth ◽  
Jin Yao ◽  
Sao-Tah Fan ◽  
Nigel Mackman
Keyword(s):  
Retinoic Acid ◽  
Tissue Factor ◽  
Human Monocytes ◽  
Monocytic Cells ◽  
All Trans Retinoic Acid ◽  
Tnf Α ◽  
Activated Monocytes ◽  
Tf Gene ◽  
Factor Α ◽  
Necrosis Factor

Expression of tissue factor (TF) by activated monocytes in several diseases leads to disseminated intravascular coagulation. Lipopolysaccharide (LPS)-induced monocyte TF expression is downregulated by the nuclear hormone all-trans retinoic acid (ATRA). In this study, we examined the mechanism by which ATRA inhibits monocyte TF expression. We show that ATRA selectively inhibited LPS induction of TF expression in human monocytes and monocytic THP-1 cells without affecting LPS induction of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8). Inhibition of TF expression occurred at the level of transcription as determined by nuclear run-on. ATRA did not significantly alter the binding or functional activity of the transcription factors c-Fos/c-Jun and c-Rel/p65, which are required for LPS induction of the TF promoter in monocytic cells. In contrast to the ATRA inhibition of the endogenous TF gene, LPS induction of the cloned TF promoter was not inhibited by ATRA in transiently transfected THP-1 cells. Our results demonstrate that ATRA selectively inhibited LPS-induced TF gene transcription in human monocytic cells by a mechanism that does not involve repression of AP-1– or NF-κB–mediated transcription.

Aging and obesity augment the stress-induced expression of tissue factor gene in the mouse

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 4011-4018 ◽  
Author(s):  
Koji Yamamoto ◽  
Takayoshi Shimokawa ◽  
Hong Yi ◽  
Ken-ichi Isobe ◽  
Tetsuhito Kojima ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Restraint Stress ◽  
Vascular Endothelial Cells ◽  
Obese Mice ◽  
Adipose Tissues ◽  
Aged Mice ◽  
Tnf Α ◽  
Underlying Mechanisms ◽  
Tf Gene ◽  
Factor Α

Hypercoagulability and thrombotic tendency are frequently induced by a variety of stressors. Clinically, aged subjects and obese patients are more susceptible to thrombotic diseases associated with stress, but the underlying mechanisms are unknown. We investigated the expression of a procoagulant gene, tissue factor (TF), in a mouse model of restraint stress. Twenty hours of restraint stress to mice caused a substantial induction of TF mRNA in several tissues. Importantly, the magnitude of induction of TF mRNA by restraint stress was larger in aged mice compared with young mice. In situ hybridization analysis of the stressed aged mice revealed that strong signals for TF mRNA were localized to renal epithelial cells, smooth muscle cells, adventitial cells, and adipocytes but not to vascular endothelial cells. These observations suggest that restraint stress induces the TF expression in a tissue-specific and cell type–specific manner. Genetically obese mice were also hyperresponsive to restraint stress in the induction of TF gene, especially in their livers and adipose tissues. Stress-induced microthrombi formation was pronounced in renal glomeruli and within the vasculature in adipose tissues of aged mice. Tumor necrosis factor-α (TNF-α) antigen in plasma was elevated by stress in aged mice and obese mice, and pretreatment of mice with anti–TNF-α antibody partially attenuated the stress-mediated induction of TF gene in adipose tissues in these mice. These results suggest that the induction of TF gene may increase the risk of stress-associated thrombosis in older and obese subjects and that TNF-α may be involved.

scholarly journals Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation

2002 ◽  
Vol 368 (2) ◽  
pp. 527-534 ◽  
Author(s):  
Zhaohua TANG ◽  
Norbert F. KÄUFER ◽  
Ren-Jang LIN
Keyword(s):  
Alternative Splicing ◽  
Protein Phosphorylation ◽  
Fission Yeast ◽  
Protein Function ◽  
Sr Proteins ◽  
Specific Protein ◽  
Glutathione S Transferase ◽  
Sr Protein ◽  
Random Screen ◽  
Related Proteins

The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1—Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast.

scholarly journals Akt2 Regulation of Cdc2-Like Kinases (Clk/Sty), Serine/Arginine-Rich (SR) Protein Phosphorylation, and Insulin-Induced Alternative Splicing of PKCβII Messenger Ribonucleic Acid

Endocrinology ◽  
2008 ◽  
Vol 150 (5) ◽  
pp. 2087-2097 ◽  
Author(s):  
Kun Jiang ◽  
Niketa A. Patel ◽  
James E. Watson ◽  
Hercules Apostolatos ◽  
Eden Kleiman ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Protein Phosphorylation ◽  
Kinase Inhibitors ◽  
Sr Proteins ◽  
Null Mouse ◽  
Phosphatidylinositol 3 Kinase ◽  
Sr Protein ◽  
Messenger Ribonucleic Acid ◽  
Extracellular Signals ◽  
Mouse Tissues

Serine/arginine-rich (SR) proteins play essential roles in the constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich (RS) domain by SR protein kinases such as Cdc2-like kinases (Clk/Sty) modulates their subcellular localization and activation. However, it remains unclear how these kinases and their target SR proteins are regulated by extracellular signals. Regulation of protein kinase C βII (PKCβII) pre-mRNA alternative splicing via exon inclusion by Akt2, a central kinase in insulin action, involves phosphorylation of SR proteins. Here we showed that Akt2, in response to insulin, resulted in phosphorylation of Clk/Sty, which then altered SR protein phosphorylation in concert with Akt2. Insulin-stimulated PKCβII pre-mRNA splicing was blocked by Clk/Sty and phosphatidylinositol-3-kinase inhibitors, and diabetic Akt2-null mouse tissues had impaired phospho-Clk/Sty, SR protein phosphorylation, and PKCβII expression. Furthermore, we observed that Akt2 phosphorylated several SR proteins distinct from Clk/Sty in response to insulin. Akt2-catalyzed phosphorylation of Clk/Sty and SR proteins revealed a role for both kinases in splicing regulation indicating dual functions for Akt2 in response to insulin in this pathway.

scholarly journals Central role of Akt in regulating Clk/Sty, serine/arginine‐rich (SR) protein phosphorylation and alternative splicing

2006 ◽  
Vol 20 (4) ◽  
Author(s):  
Denise R. Cooper ◽  
Karen D. Corbin ◽  
James E. Watson ◽  
Masatoshi Hagiwara ◽  
Niketa A. Patel ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Protein Phosphorylation ◽  
Sr Protein

scholarly journals Heme induces mRNA expression and activation of tissue factor by TLR4 dependent mechanisms

2020 ◽  
Author(s):  
B.W. Hounkpe ◽  
C.R.P. Moraes ◽  
M.N.N. do Santos ◽  
F. F. Costa ◽  
E.V. De Paula
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Mononuclear Cells ◽  
Cell Disease ◽  
Coagulation Activation ◽  
Coagulation Activity ◽  
Human Mononuclear Cells ◽  
Tnf Α ◽  
Dependent Pathway

AbstractIntroductionHemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. Evidence showing that heme can induce tissue factor (TF) expression in endothelial cells and TF-dependent coagulation activation in animal models of SCD suggest that heme can contribute to hypercoagulability in this condition. We recently demonstrated that heme can induce coagulation activation in whole blood of healthy volunteers in a TF-dependent fashion.MethodsHerein, we aimed to evaluate whether this heme-induced coagulation activity was dependent on the expression and/or activation of hematopoietic TF in human mononuclear cells. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of FXa.ResultsHeme was capable of inducing TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming.ConclusionOur results provide additional evidences on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.

KLF11 Protects against Venous Thrombosis via Suppressing Tissue Factor Expression

2021 ◽  
Author(s):  
Wenying Liang ◽  
Haocheng Lu ◽  
Jinjian Sun ◽  
Guizhen Zhao ◽  
Huilun Wang ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Tissue Factor ◽  
Gene Transcription ◽  
Loss Of Function ◽  
Expression Change ◽  
Causative Gene ◽  
Tnf Α ◽  
Gene Assay ◽  
Tf Gene

AbstractKrüppel-like factors (KLFs) play essential roles in multiple biological functions, including maintaining vascular homeostasis. KLF11, a causative gene for maturity-onset diabetes of the young type 7, inhibits endothelial activation and protects against stroke. However, the role of KLF11 in venous thrombosis remains to be explored. Utilizing stasis-induced murine deep vein thrombosis (DVT) model and cultured endothelial cells (ECs), we identified an increase of KLF11 expression under prothrombotic conditions both in vivo and in vitro. The expression change of thrombosis-related genes was determined by utilizing gain- and loss-of-function approaches to alter KLF11 expression in ECs. Among these genes, KLF11 significantly downregulated tumor necrosis factor-α (TNF-α)-induced tissue factor (TF) gene transcription. Using reporter gene assay, chromatin immunoprecipitation assay, and co-immunoprecipitation, we revealed that KLF11 could reduce TNF-α-induced binding of early growth response 1 (EGR1) to TF gene promoter in ECs. In addition, we demonstrated that conventional Klf11 knockout mice were more susceptible to developing stasis-induced DVT. These results suggest that under prothrombotic conditions, KLF11 downregulates TF gene transcription via inhibition of EGR1 in ECs. In conclusion, KLF11 protects against venous thrombosis, constituting a potential molecular target for treating thrombosis.

Adiponectin inhibits tissue factor expression and enhances tissue factor pathway inhibitor expression in human endothelial cells

2008 ◽  
Vol 100 (08) ◽  
pp. 291-300 ◽  
Author(s):  
Yi-Jian Chen ◽  
Li-Qun Zhang ◽  
Guang-Ping Wang ◽  
Hui Zeng ◽  
Ben Lü ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Tnf Α ◽  
Tissue Factor Pathway ◽  
Expression And Activity

SummaryTissue factor (TF) plays a pivotal role in thrombus formation and atherogenesis in acute coronary syndrome. Tissue factor pathway inhibitor (TFPI) is a specific physiological inhibitor of TF/ FVIIa complex that regulates TF-induced coagulation. Adiponectin (Adp) is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Adp inhibits inflammatory cytokine and adhesion molecules expression, and it can prevent endothelial dysfunction. In this study, we investigated the effects of Adp on tumor necrosis factor-α (TNF-α)-induced expression of TF and TFPI in human umbilical vein endothelial cells (HU-VECs), and the signaling transduction pathways involved. It was found that Adp significantly inhibited both TF protein expression and activity in TNF-α-stimulated HUVECs. In the meanwhile, it increased TFPI protein expression and activity for about two folds. Adp also inhibited TF mRNA expression induced by TNF-α, but had no effect on TFPI mRNA expression. The inhibitory effect of Adp onTNF-α-inducedTF expression was prevented by pretreatment with Rp-cAMPs, a PKA inhibitor. Adp increased intracellular cAMP content and PKA activity levels in a dose-dependent manner. Phosphorylation of IκB-α was decreased by Adp, but phosphorylation of p44/42MAPK, SAPK/ JNK, and p38MAPK were not affected. These results suggested that Adp inhibits TF expression through inhibition of a PKA dependent nuclear factor- κB (NF-κB) signaling pathway. It was also found that adiponectin promoted Akt and AMP-activated protein kinase phosphorylation. The inhibitory effect of Adp on TNF-α-induced TF synthesis was abrogated in part by pretreatment with the PI3kinase inhibitor LY 294002, suggesting that Akt activation might inhibit TF expression induced by TNF-α. The inhibitory effect of Adp is almost completely abrogated by inhibition of both the cAMP/PKA pathway and PI3K/Akt pathway. In conclusion, our data indicated that inhibition of NF-κB through stabilization of IκB-α and activation of Akt phosphorylation may mediate the inhibitory effect of Adp on TF expression; but the enhancement effect of Adp on the TFPI production might occur via translational rather than transcriptional regulation.

Sign in / Sign up

Export Citation Format

Share Document