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Keywords:

  • fibrinolysis;
  • gene regulation;
  • inflammation;
  • mRNA stability;
  • transcription

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

Summary.  The maintenance of a given physiological process demands a coordinated and spatially regulated pattern of gene regulation. This applies to genes encoding components of enzyme cascades, including those of the plasminogen activating system. This family of proteases is vital to fibrinolysis and dysregulation of the expression pattern of one or more of these proteins in response to inflammatory events can impact on hemostasis. Gene regulation occurs on many levels, and it is apparent that the genes encoding the plasminogen activator (fibrinolytic) proteins are subject to both direct transcriptional control and significant post-transcriptional mechanisms. It is now clear that perturbation of these genes at either of these levels can dramatically alter expression levels and have a direct impact on the host’s response to a variety of physiological and pharmacological challenges. Inflammatory processes are well known to impact on the fibrinolytic system and to promote thrombosis, cancer and diabetes. This review discusses how inflammatory and other signals affect the transcriptional and post-transcriptional expression patterns of this system, and how this modulates fibrinolysis in vivo.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

The host’s fibrinolytic capacity is provided by the plasminogen activator family of proteins. This family consists of a number of proteases and protease inhibitors that regulate the conversion of plasminogen to its active form, plasmin [1]. Although plasmin is regarded as the effector arm of this cascade, recent advances in the field have shown that the individual components of this system can influence cell physiology in their own right. Indeed, the influence of the plasminogen activating system extends beyond its fibrinolytic capacity to remove blood clots.

There are two major plasminogen activators, tissue-type and urokinase-type plasminogen activator (t-PA and u-PA, respectively), that are structurally related and are expressed in many cell types and tissues. The proteolytic activity of these two plasminogen activators is inhibited by plasminogen activator inhibitor (PAI) type-1 and PAI-2, while plasmin activity is controlled by alpha2 antiplasmin. Like their protease targets, both PAI-1 and PAI-2 are highly regulated genes that can be induced many-fold by various mediators. Another key component of this family is the urokinase receptor (u-PAR). u-PAR is a GPI-linked cell surface receptor that not only facilitates u-PA dependent proteolytic activity to the cell surface, but can also initiate intracellular signaling [2].

Linkages between coagulation, fibrinolysis, and inflammation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

Although the fibrinolytic and coagulation systems perform opposite functions, regulation between these enzyme cascades is critical to prevent inappropriate clot formation or its removal. Also, there is a growing body of evidence that now links fibrinolysis and coagulation pathways with inflammation [3]. An example of an interaction between these systems is provided by thrombin [4] and ‘thrombin activatable fibrinolysis inhibitor’ (TAFI) [5]. In the presence of thrombomodulin (TM), thrombin activates the anticoagulation pathway [6] and TAFI, a carboxypeptidase that inhibits fibrinolysis [7]. Activated TAFI has anti-inflammatory actions by virtue of its ability to inactivate the complement component C5a [8,9]. In another example, endotoxin-induced tumor necrosis factor (TNF) production is inhibited by PAI-1 [10] while TNF, a potent inflammatory mediator, markedly suppresses fibrinolytic activity. The linkage between fibrinolysis and inflammation has also been provided from studies using fibrinogen −/− mice. These mice showed reduced macrophage adhesion and lower cytokine production during inflammation [11]. Taken together, these examples clearly illustrate the interconnections between coagulation, fibrinolysis and inflammation.

Inflammatory cytokines and fibrinolysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

Although TNF can modulate expression levels of essentially all of the major components of the fibrinolytic system, its profound stimulatory effect on PAI-1 and down-regulation of t-PA expression in endothelial cells are arguably the most significant. In patients with septic shock, elevated levels of TNF are correlated with a substantial reduction in levels of t-PA antigen [12]. Inhibition of TNF in plasma has also been shown to down-regulate PAI-1 levels [13]. More recently, a polymorphism in the TNF receptor 1 gene has been correlated with PAI-1 levels in obese women [14]. Hence, TNF is clearly a (patho)-physiological regulator of the fibrinolytic response. Many laboratories have therefore devoted much effort to elucidate the molecular mechanisms by which TNF and other inflammatory cytokines regulate the expression of the fibrinolytic components and this review highlights some recent advances in the field.

Regulation of gene expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

The extent to which any protein is expressed in a cell is largely dependent on the relative abundance of its mRNA at any given time. The cellular abundance of mRNA is influenced not only by direct transcriptional activity but also by its decay rate. Genetic studies have highlighted the importance of studying gene expression patterns. Indeed, evidence supporting a clinical consequence of dysregulated gene expression in hemostasis has been derived from genetic studies in which polymorphisms in key regulatory elements within gene promoters (i.e. the 4 G/5 G and −7351 C/T polymorphisms in the PAI-1 [15] and t-PA genes [16], respectively) or within transcripts (i.e. the G20210A polymorphism in the prothrombin 3′-UTR [17]) have resulted in altered expression that correlates with clinical outcome.

Transcriptional and post-transcriptional regulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

Transcriptional processes are modulated by signature motifs within gene promoters and occasionally elsewhere within the structural gene. Many laboratories in the field of thrombosis and fibrinolysis strived to identify regulatory elements and associated transcription factors that were critical in the response of specific genes to a given stimulus and the signaling pathways that were driving these events. With respect to post-transcriptional regulation, most studies published to date have addressed changes in mRNA stability. Changes in the decay rate of mRNA have emerged as an important component of plasminogen activator gene regulation that is itself a highly regulated process [18]. Generally speaking, mRNA stability is a property of the 3′untranslated region (3′-UTR) of the transcript, and akin to the search for regulatory elements in gene promoters, 3′-UTRs have been screened for cis-acting elements and mRNA binding proteins that are important in this process.

The best described elements involved in mRNA stability are adenylate and uridylate (AU)-rich elements (ARE), first described in 1986 [19], that usually harbor repeats of AUUUA or UUAUUUAUU [20]. The means by which an ARE can promote mRNA decay is the subject of intense research, although one major mRNA turnover pathway in mammalian cells involves deadenylation as the initial event that triggers the subsequent decapping and decay of the mRNA body [21,22].

Another consideration is that changes in gene transcription and mRNA decay are occasionally coupled. Hence to understand more fully the means by which a given agonist alters the pattern of gene expression, it is important to address both transcriptional and post-transcriptional parameters. The genes of the fibrinolytic system are no exception to this and the impact of both transcription and post-transcriptional control of these genes by growth factors and agents associated with inflammation will be highlighted.

PAI-1

Elevated levels of plasma PAI-1 have been found in several pathologic conditions and have been linked to an increase in the risk for vascular complications such as thrombosis [23], myocardial infarction [24] and obesity [25]. PAI-1 is expressed in almost all cell types, including adipocytes, hepatocytes and endothelial cells. Infusion of TNF into mice has been shown to increase PAI-1 expression in adipocytes [26]. PAI-1 is also regulated by transforming growth factor beta (TGF-B), dexamethasone, and hypoxia (see [27]). More recently, PAI-1 expression was shown to be induced in macrophages by the complement component C5a [28].

The response of the PAI-1 gene to TGF-β and TNF is quite profound. TGF-β mediated PAI-1 transcription is mediated by binding of SMAD 3 and 4 proteins to consensus sites in the PAI-1 promoter [29], which is further associated with Ras MEK and p38 MAP kinase pathways for inducible expression [30]. Other signature motifs include Sp1 elements in the proximal promoter that are needed in response to glucose [31] and angiotensin II [32] signaling while an AP-1-like binding site mediates the PAI-1 response to protein kinase (PK)-C and PK-A [33,34]. PAI-1 is also induced following hypoxia and a hypoxia response element (HRE) has been identified [35].

Despite the fact that the PAI-1 gene is powerfully regulated by TNF, the means by which TNF promotes PAI-1 transcription has been elusive. TNF regulation is often associated with activation of NF-κB and its binding to NF-κB consensus elements. However, initial reports found no evidence for NF-κB sites in the PAI-1 promoter. In one report, such an element was dismissed as TNF appeared to be acting via a Nur77 binding site in the proximal promoter region [36]. To further support this contention, increased expression of Nur77 in co-transfection assays was correlated with increased PAI-1 expression. However, a subsequent study indeed revealed the presence of a conserved NF-κB element within the promoter, yet this was located far upstream (∼ 15 kb) from the transcription start site [37]. Furthermore, this distal element was shown to provide a binding site for the NFκB subunit p50 and p65 in vitro.

A recent investigation into the mechanisms underlying transcriptional induction of the PAI-1 gene by TNF, insulin and TGB-β in adipocytes showed the requirement for the PKC pathway [26]. More recently, E2F was revealed as a critical downstream mediator in 3T3 L1 pre-adipocytes with a decisive role in regulating PAI-1 gene expression [38].

While many cis-acting elements have been identified in the PAI-1 promoter, the 4 G/5 G polymorphism at position −675 [15] has been correlated with the incidence of thrombosis. This single nucleotide substitution alters the rate of PAI-1 transcription in vitro, and the alteration in plasma PAI-1 levels has been associated with the development of cerebral ischemia [39], myocardial infarction [15], and obesity [40]. An interesting question is whether this functional polymorphism influences the response of the PAI-1 gene to cytokines and other stimulatory agents. Evidence to date, however, suggests that 4 G/5 G does not influence the response of the PAI-1 gene to growth factors and cytokines, as endogenous PAI-1 levels in cultures of human arterial smooth muscle cells (genotyped for 4 G/4 G, 4 G/5 G or 5 G/5 G) responded similarly to these stimulants [41].

Post-transcriptional control of PAI-1

In humans, there are two PAI-1 mRNA transcripts (3.2 kb and 2.2 kb). These two transcripts are expressed in a tissue-specific manner and are regulated differently [42–44] and produced as a result of alternative cleavage and polyadenylation sites within the PAI-1 gene [45]. The functional significance of the two transcripts in humans has been a long-standing question in this field and still remains to be elucidated.

The 3.2.kb PAI-1 transcript is composed of a long 3′-UTR with several potential AU-rich elements [46]. This form is less stable, most likely due to the presence of a copy of the pentamer (AUUUA), which is absent from the more stable 2.2 kb form (half-life 2.5–2.8 h) [42]. The presence or absence of this cis-element between the two mRNA species therefore gives rise to variations in the post-transcriptional regulation of PAI-1.

In Hep G2 hepatoma cells, TGF-β and insulin increase the half-life of 3.2 kb PAI-1 mRNA but not the 2.2 kb form [47], whereas insulin-like growth factor stabilizes both species of PAI-1 mRNA [43,47].

The best-studied example of post-transcriptional control of PAI-1 is by cAMP. cAMP analogs decrease PAI-1 mRNA levels in HTC rat hepatoma cells [48], while the PAI-1 3′-UTR was able to confer a cyclic nucleotide dependent instability to a reporter gene. This instability element, identified as the PAI-1 cAMP responsive sequence (PAI-CRS) [49], was unrelated to the classical AU-rich sequence [50].

There are no reports currently to hand as to whether TNF has the capacity to alter PAI-1 mRNA decay. This would be a worthwhile endeavor as it remains to be determined whether the transcriptional response of PAI-1 to TNF fully accounts for the complete magnitude of this induction.

PAI-2: its role and regulation during inflammation

PAI-2 was once considered the ‘enigmatic’ serpin and for good reason [51] as the major part of PAI-2 exists as a non-glycosylated intracellular protein [52] well hidden from its target u-PA. Increasing evidence now supports a role for PAI-2 as a modulator of intracellular proteolytic events associated with cell cycle control [53], cancer cell survival [54], cell proliferation [55], gene transcription [56] and, most recently, with inflammation and the innate immune response [57]. For the latter, PAI-2 was identified as a key survival gene in response to Bacillus anthracis infection. Evidence has also accumulated that links PAI-2 with apoptosis [58–60], although other reports have disputed this [61].

The PAI-2 gene is one of the most highly regulated genes known. In fibroblasts and monocytic cell systems, PAI-2 expression is markedly up-regulated by TNF and endotoxin (LPS). Indeed, the PAI-2 (and PAI-1) genes were the first genes shown to be directly responsive to TNF at the transcriptional level [62]. PAI-2 is also up-regulated by agents that promote cellular differentiation [63]. Differential gene expression profiling (SAGE) of LPS-treated primary human monocytes identified PAI-2 as the third most inducible gene, being induced 105-fold by this agent [64], consistent with a role for PAI-2 in the innate immune response [57]. In a microarray study to identify genes influenced by elevated levels of serum lipoprotein(a) (Lp(a)) in human monocytes, PAI-2 mRNA was the most induced transcript [65]. Curiously, the induction of PAI-2 to Lp(a) was gender specific, with the effect only seen in monocytes from males.

Although various agents control PAI-2 gene expression [66], the most investigated are tumor promoters, including phorbol esters (i.e. PMA) and TNF. At the transcriptional level, two important AP1-like elements reside in the proximal PAI-2 promoter, AP1a and AP1b, and one CRE-like element [67]. Promoter deletion analyses of the PAI-2 promoter following TNF treatment in HT-1080 cells showed TNF inducibility only after the promoter was truncated to −219 [68], indicating the presence of a TNF repressor region upstream. Antalis et al. [69] characterized 5.1 kb of the PAI-2 promoter in U937 cells by deletion analysis and identified another silencer between −1977 and −1675 termed ‘PAI-2-upstream silencer element-1’ (PAUSE-1), but its role in the regulation of PAI-2 by inflammatory stimulants was not explored.

PAI-2 was identified by Park et al. as a macrophage survival factor following a screen of LPS-inducible NF-κB target genes that also required p38 activity [57]. The NF-κB target gene was in fact CREB, but the downstream target of CREB was found to be PAI-2. Indeed, the induction of PAI-2 by LPS was blocked if CREB phosphorylation was inhibited. It was suggested that a putative NF-κB binding site at −860 and a CREB binding site at −1319 were the functional sites in the PAI-2 promoter, but this was not demonstrated directly.

Post-transcriptional control of PAI-2 expression

The extent of transcriptional activation of the PAI-2 gene to some agonists does not always correlate with the level of mRNA accumulation [70], while more direct studies using transcription blockers even showed that suppression of PAI-2 mRNA by dexamethasone involved acceleration in the decay rate of the PAI-2 transcript [71]. Collectively, these findings implicated alteration in PAI-2 mRNA stability in both induction and down-regulation of the PAI-2 gene.

PAI-2 mRNA contains a 580 nt long 3′UTR [72] that harbors a functional ARE element [70,73]. This element also provides binding sites for several ARE binding proteins, including the stabilizing protein HuR and the potent mRNA destabilizing protein, tristetraprolin (TTP) [74]. Overexpression of TTP in HEK 293 cells transfected with a constitutively active PAI-2 expression vector resulted in loss of PAI-2 mRNA, suggesting that TTP indeed regulated PAI-2 expression. It has yet to be shown directly if TNF-inducible expression of PAI-2 involves changes in ARE-dependent mRNA decay. It is curious to point out that some of the PAI-2 mRNA binding proteins identified are themselves highly TNF-responsive [75].

Urokinase

Urokinase-type plasminogen activator (uPA) is a multifunctional molecule that serves either as a proteolytic enzyme or as a signal-inducing ligand. uPA has been shown to play an important role in the cell-mediated immune response, in part by the generation of pro- and anti-inflammatory signals [76], to influence immune-complex mediated inflammation [77] and to be regulated by inflammatory mediators including TNF in endothelial cells [78] and keratinocytes [79].

u-PA is also commonly associated with extravascular proteolytic events, including wound healing, and under pathological conditions in the metastatic spread of tumors. Using transgenic models in which the u-PA gene is overexpressed in macrophages, evidence has accumulated to implicate a pathological role for u-PA in the development of cardiac fibrosis [80] in a process that was dependent on the presence of plasminogen [81]. Further studies along these lines also implicated u-PA in atherosclerosis and coronary artery occlusions [82]. u-PA has also been implicated as a modulator of vasoconstriction during atherosclerosis [83].

Transcriptional regulation of u-PA expression

u-PA was one of the first genes of the fibrinolytic system to be studied at the transcriptional level [84]. The u-PA gene promoter harbors a collection of regulatory elements, with many proving to be functionally relevant [85,86]. Promoter deletion analyses have revealed a number of functionally important regulatory domains [27] and many signaling pathways have been identified. Transcription of the human u-PA gene is modulated by an inducible enhancer located at −2.0 kb [87]. This activity requires the cooperation of an upstream composite Ets/AP1a and a downstream AP1b site. These elements are highly conserved, suggesting their importance in u-PA gene regulation [88]. These Ets/AP1 sites in the u-PA promoter are the final target elements of the signaling cascades induced by PMA [88–90], growth factors [91–94], and oncogenes [91,95,96]. The u-PA gene also has a functional NFκB-like sequence at −1583 bp that was shown to mediate PMA induction in HeLa and HepG2 cells via formation of two NF-κB heterodimeric complexes, one consisting of Rel/p65 and the other of p65/p50 [97].

Post-transcriptional control of u-PA expression

The 3′-UTR of u-PA contains two highly conserved mRNA instability determinants: a classical ARE and another site that acts independently [98]. It is interesting that the degree of u-PA mRNA stability is linked to its expression levels in metastatic cells [99,100]. More recently, Tran et al. [101] identified three u-PA mRNA binding proteins in HeLa nuclear extracts: two are known ARE-binding proteins, HuR and NFAR, and the putative DExH RNA helicase, RHAU. These findings are relatively new and it remains to be determined how these mRNA binding proteins alter the expression pattern of the u-PA gene in cells following cell stimulation by growth factors or inflammatory signals.

The u-PA receptor

Since its initial discovery in 1989 [102] and cloning in 1990 [103], the u-PA receptor (u-PAR) has proven to be an integral component of the plasminogen activator cascade. uPAR expression is associated with cancer progression [2], and also with inflammation. uPAR levels are elevated in inflammatory conditions, including rheumatoid arthritis [104] and glomerulonephritis [105]. Also, uPAR −/− mice have higher cytokine and chemokine levels and a higher bacterial load during pyelonephritis [106], indicating a role for uPA during infection.

uPAR expression is modulated by cytokines and growth factors [107–109]. TGFβ, for example, increases u-PAR transcription [110], while PMA induces u-PAR expression in many tumor cell lines, consistent with a role for u-PAR expression in the metastatic spread of tumors.

u-PAR promoter analyses identified a functional element between −141 and +47 relative to the transcription initiation site [111]. The promoter region lacks conventional TATA and CAAT boxes but contains a CpG-rich island and sequences related to consensus cis-acting elements for AP1, AP2, NF-κB and Sp1 that mediate the basal transcription of the gene. A GC-rich region between −99 and −70 that recognized Sp1, was required for basal expression of the gene [111]. A separate sequence at −148/−124 was also identified that could bind Sp1/Sp3 and an AP2α-related factor and was required for basal and PMA-inducible u-PAR expression in colon cancer cells [112]. Hapke et al. [113] identified a silencing motif consisting of a PEA3/Ets sequence at −248 bp that mediated the down-regulation of u-PAR expression by integrin β3. Recently, u-PAR expression has been linked to the expression and ligation of αvβ3 [114]. An enhancer element has also been identified within the first intron of the u-PAR gene that is necessary for both basal and inducible expression [115].

Post-transcriptional regulation of u-PAR

Phorbol ester and TGFβ-mediated increase in u-PAR mRNA levels in A459 cells is associated with an increase in the stability of the u-PAR transcript [116]. A u-PAR mRNA binding protein identified as phosphoglycerate kinase (PGK) [117] was shown to bind to a 51-nt destabilizing element within the u-PAR coding region [118]. The 3′UTR of u-PAR mRNA also harbors a functional ARE of approximately 50 nt in length [119]. Although this ARE confers instability, in human Jurket T cells this is overcome by the engagement of the β2-integrin LFA-1 [119]. In addition, the mRNA stabilizing protein, HuR, specifically interacts with the u-PAR ARE and overexpression of HuR stabilizes the u-PAR mRNA in HeLa cells [120]. In u-PAR-transfected kidney cells, u-PA increased u-PAR expression at a post-transcriptional level, by increasing the activity of a novel cellular factor that binds the coding region instability determinant of u-PAR mRNA, that is presumably stabilizing the transcript [121].

Tissue-type plasminogen activator

t-PA is best known for its role as the primary endogenous intravascular fibrinolytic protease, and its expression and regulation in endothelial cells has been the topic of much research. However, the fact that t-PA is expressed in extravascular regions [122–124] suggests that the role of t-PA in vivo is more diverse than initially thought. t-PA has also been shown to influence the inflammatory response [125]. In this study, t-PA was shown to have a protective function in abdominal sepsis caused by Escherichia coli in a manner that was independent of plasmin generation. t-PA also modulates inflammatory responses and renal function in ischemia reperfusion injury [126]. In this report, tPA−/− mice were shown to have a reduction of ischemia-induced inflammation and had reduced neutrophil influx.

t-PA gene expression is modulated by a variety of cytokines and growth factors. It is activated by growth factors (i.e. EGF) and cAMP [127]. Steroid hormones and 1,25-dihydroxyvitamin D3 increased t-PA synthesis in vivo and in vitro [128,129], while retinoic acid induced t-PA expression in microvascular endothelial, oral squamous carcinoma and neuroblastoma cells [130]. In the context of inflammatory signals, t-PA expression is potently down-regulated by TNF [62,131]. When coupling the suppressive effect of TNF on t-PA with its ability to induce PAI-1 strongly, one can easily understand why TNF is such a potent anti-fibrinolytic agent. t-PA is also regulated in a cell type-specific manner. For example, PMA transcriptionally up-regulates the t-PA gene in HeLa cells [132] and in endothelial cells [133] but in HT-1080 cells, PMA suppresses t-PA expression [63].

Functional analysis of the proximal human t-PA promoter identified two critically important elements: a CRE-like sequence and a GC box (Sp-1 binding site) that cooperated during constitutive and PMA-induced transcription in HeLa cells [132,134] and in endothelial cells [135]. A novel upstream NF-κB site was recently identified that was shown to influence PMA-mediated induction of t-PA in neuronal-like cells [136]. Interestingly, differential binding of CREB-1 and ATF2 to the human AP1-like element appears to correlate with the differential regulation of t-PA by phorbol esters in HT-1080 and HeLa cells [134]. Besides Sp1, a novel GC box-binding protein has been suggested to be responsible for neuronal-specific t-PA gene expression [137]. These sites have also been verified by in vivo footprinting analysis in different cell lines [135,136]. A second important domain within the t-PA promoter was described by Bulens et al. [128], who identified a multi-hormonal responsive region between −7.1 and −8.0 kb that has been shown to be a unique enhancer necessary for hormone activation.

The mechanism by which TNF down-regulates the t-PA gene at the transcriptional level has only recently been brought to light. Ulfhammer et al. [138] showed that TNF-mediated suppression of t-PA was dependent on NF-κB activation, and also on p38 MAPK signaling. Evidence was also provided for TNF-induced binding of proteins to the novel upstream NF-κB element [136] and to the proximal CRE element.

Recent data have shown that even subtle alterations in t-PA transcription in vivo can have pathophysiological consequences for fibrinolysis. For example, a single nucleotide polymorphism (C/T) at position −7351 in the t-PA promoter is associated with lower plasma t-PA levels and an increased risk of developing myocardial infarction [16]. This polymorphism also directly alters the binding affinity of Sp1 to this polymorphic region [139], providing, at least in part, a mechanistic basis for the change in t-PA expression. More recently, evidence has been provided for an involvement of this polymorphism in regulated expression of t-PA in endothelial cells by PMA [140], thereby implicating this polymorphism in a broader context of t-PA gene regulation.

Post-transcriptional control of t-PA

Ouyang et al. [141] showed that deletion of the entire 5′-UTR from t-PA mRNA resulted in an increase in t-PA mRNA stability in transfected COS cells. The influence of the 5′-UTR is interesting because the t-PA gene has two transcription initiation sites (TIS) 110 bp apart [142]. The experiments by Ouyang et al. were based on the upstream TIS, which produces a 5′-UTR of 209 nt. In two human cell systems (WI-38 fibroblasts and endothelial cells), the second TIS in fact is preferred [133,142], creating a 5′-UTR of only 99 nt, and it is possible that the two t-PA transcripts may decay at different rates due to the different lengths of the 5′-UTRs.

The 3′-UTR of t-PA mRNA lacks classical AU-rich elements, yet deletion of the entire 3′-UTR causes a three-fold increase in t-PA mRNA stability in transfected COS cells [143]. t-PA expression is also regulated developmentally at the translational level [144]. Translational activation of t-PA mRNA during meiotic maturation is associated with poly(A) tail elongation [145,146] in a process involving an AU-rich element within the 3′-UTR of t-PA mRNA [147]. More recently, a cytoplasmic polyadenylation element was shown to exist within the 3′-UTR that was responsible for directing mRNA degradation during embryonic development [148].

TAFI

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

TAFI, a plasma zymogen that is activated by thrombin, is the terminal enzyme of the coagulation cascade [149]. Activation of TAFI by thrombin is accelerated over 1000-fold in the presence of the endothelial cell membrane protein TM [150]. Activated TAFI (TAFIa) also removes the carboxyl-terminal arginine residues from C3a and C5a [8,9], thus implying a role for the TAFI pathway in the vascular responses to inflammation. Injection of mice with bacterial lipopolysaccharide results in an increased hepatic TAFI expression [151] and it was suggested that TAFI was an acute phase protein. However, in studies on patients with sepsis, TAFI levels were found to be decreased [152].

TAFI gene expression

Only a limited number of studies investigated the molecular mechanisms controlling TAFI expression. TAFI is not a highly regulated gene. Nonetheless, treatment of HepG2 cells with a combination of interleukin (IL)-1 and IL-6 (but not alone) lowers TAFI mRNA levels in HepG2 cells and is correlated with a decrease in TAFI mRNA stability [153,154]. This same group also showed that dexamethasone increased TAFI mRNA levels and gene promoter activity. TAFI expression has also been shown to be up-regulated in adipocytes by insulin [155].

The gene encoding TAFI has been characterized relatively recently [156]. Many polymorphisms have been identified throughout the TAFI gene, including the 5′-flanking region and the 3′-UTR. Some of these polymorphisms are associated with variations in plasma TAFI levels [157,158]. The TAFI promoter lacks a consensus TATA box and transcription was shown to be initiated from multiple sites. Promoter deletion analyses also revealed a role for a 73 bp region within the proximal region that was necessary for basal transcription in HepG2 cells. Subsequent studies identified a putative C/EBP-binding site that influenced promoter activity in transfected HepG2 cells [159]. TAFI mRNA exists in three forms, each a consequence of alternate use of one of three polyadenylation sites in the TAFI 3′-UTR resulting in TAFI transcripts with 3′-UTRs of 390, 423 or 549 nucleotides long. These transcripts each display different intrinsic stabilities, with the shorter variant having the longest half-life. A particularly novel finding was that treatment of HepG2 cells with IL-1 and IL-6 resulted in a marked change in the relative abundances of the three TAFI mRNA variants [154]. The outcome of this was that the longer transcript was preferentially expressed.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

The formation and degradation of a blood clot is an important aspect of the inflammatory response. The genes encoding the major components of the fibrinolytic system are regulated to varying degrees by inflammatory mediators and a collection of growth factors and other stimulants. It is also apparent that the same inflammatory agonist can simultaneously influence expression of many of the fibrinolytic genes. With closer scrutiny of the mechanisms underlying these effects, we are beginning to appreciate the transcriptional and post-transcriptional processes that are activated by these stimulants. One of the themes of this review has been the interrelationship between inflammation, coagulation and fibrinolysis and how this is manifested at the level of gene expression. It is clear that the host has to deal with a fine regulatory balance between protection and pathology when faced with the coordinated activation of these systems.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References

The author would like to thank Y. Nagamine and A. Samson for advice and for reading the manuscript. This work was supported by grants to R.L.M. from the National Health and Medical Research Council of Australia. The author is indebted to all of the members of the Medcalf laboratory who over the years have contributed to various aspects of plasminogen activator biology and gene regulation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Linkages between coagulation, fibrinolysis, and inflammation
  5. Inflammatory cytokines and fibrinolysis
  6. Regulation of gene expression
  7. Transcriptional and post-transcriptional regulation
  8. TAFI
  9. Conclusion
  10. Acknowledgments
  11. Disclosure of Conflict of Interests
  12. References
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