The early in-vivo effects of a single anti-emetic dose of dexamethasone on innate immune cell gene expression and activation in healthy volunteers.

Dexamethasone is often administered to surgical patients for anti-emetic prophylaxis. This study examined the early (up to 24 h) in-vivo effects of dexamethasone (8 mg) to demonstrate the magnitude and temporal nature of changes on circulating peripheral blood mononuclear cell gene expression and activation in 10 healthy male volunteers. Blood samples were drawn at baseline, 2 h, 4 h and 24 h. Gene expression was measured using quantitative real-time polymerase chain reaction. Cytokine expression was measured using multiplex immuno-assays. Innate immune cell phenotypes were examined with flow cytometry. Dexamethasone resulted in rapid transient changes in immunophilin (p = 0.0247), plasminogen activator inhibitor-1 (p = 0.0004), forkhead box P3 (p = 0.0068) and dual specific phosphatase-1 (p = 0.0157) gene expression at 4 h compared with pre-dexamethasone. Plasma interleukin-10 levels increased within 2 h (p = 0.0071) and returned to baseline at 24 h. Reductions in classical (p = 0.0009) and intermediate monocytes (p = 0.0178) and dendritic cells (p = 0.0012) were followed by increases in the level of these populations at 24 h compared with pre-dexamethasone (classical monocytes p = 0.0073, intermediate monocytes p = 0.0271, dendritic cells p = 0.0142). There was a profound reduction in the mean fluorescence intensity of the maturation marker, human histocompatibility leucocyte antigen, at 24 h in all monocyte subsets (p = 0.0002 for classical and non-classical monocytes, p = 0.0001 for intermediate monocytes) and dendritic cells (p = 0.0001). This study confirms rapid transient effects of 8 mg dexamethasone on innate immune cells with the potential to alter the inflammatory response to surgery and provides support for the hypothesis that intra-operative administration may be both immunosuppressive and immune-activating in the immediate peri-operative period.

A flowcytometric analysis to efficiently quantify multiple innate immune cells and T Cell subsets in human blood.

The balance of inflammation and immunosuppression driven by changed ratios in diverse myeloid and T cell subsets, as well as their state of activation and ability to migrate to lymphoid compartments or inflammatory sites, has emerged as a highly active area of study across clinical trials of vaccines and therapies against cancer, trauma, as well as autoimmune and infectious diseases. There is a need for effective protocols which maximally use the possibilities offered by modern flow cytometers to characterize such immune cell changes in peripheral blood using small volumes of human blood. Additionally, longitudinal clinical studies often use cryopreserved samples, which can impact flow cytometric results. To efficiently gauge both the innate and the adaptive immune response, two novel 15-color antibody panels to identify key myeloid and T cell subsets and their functional potential were established. This approach was used to compare cellular immune profiles in fresh whole blood and in matched cryopreserved peripheral blood mononuclear cells (PBMCs). Cocktail I was designed to identify and characterize myeloid cell populations including dendritic cells (DCs), monocytic monocyte-derived suppressor cells (MO-MDSC), and monocytes, determining further core aspects of their state of maturity, T cell stimulatory (or inhibitory) potential, and migration capability. Cocktail II was used for phenotyping diverse T cells subsets, and their key migration and functional regulatory capabilities. The two 15-color antibody panels for the evaluation of both immune-stimulating and immunosuppressive processes presented herein allowed for efficient evaluation of the balance of immune activation versus immunosuppression across key blood cells, with good resolution for all 15 markers stained for in each panel. Gating strategies for the myeloid and T cells are presented to further support specific subset identification. This protocol was shown to be reproducible across donors and useful to study both RBC-lysed whole blood and cryopreserved PBMCs. © 2017 International Society for Advancement of Cytometry.

Evaluation of gait impairment in mice subjected to craniotomy and traumatic brain injury.

Traumatic brain injury (TBI) represents a significant global health burden and causes long-lasting neuromotor deficits, particularly in individuals who sustain severe TBI. A better understanding of gait impairment after experimental TBI will provide valuable information for the recovery and rehabilitation of TBI survivors. Here we utilised the DigiGait system to perform kinematic gait analysis in mice subjected to brain injury induced by the controlled cortical impact (CCI) TBI model. Naïve mice, non-craniotomised and craniotomised mice were included as controls. The temporal and spatial profile of gait was mapped from 3h to 1-week post-TBI. Remarkably, there was a noticeable alteration in some aspects of gait in craniotomised sham mice from their pre-surgery baseline at various time-points over the testing period. This was not observed in naïve mice or non-craniotomised sham controls over the same time period. This finding indicates that the craniotomy procedure alone effects gait. When craniotomised mice were subjected to TBI, additional deleterious effects on gait function were observed, including forelimb stance and swing duration as well as left hindlimb swing and stride duration and frequency. Hence, mice subjected to CCI-induced TBI develop clear alterations in gait but part of this is attributable to the effect of craniotomy alone. This study also highlights the need to include both non-craniotomised and craniotomised sham mice as controls when undertaking the CCI-induced model of TBI, particularly when early time points are being evaluated.

Activated platelets rescue apoptotic cells via paracrine activation of EGFR and DNA-dependent protein kinase.

Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK--an endogenous cytoprotective cascade.

Fibrinolysis, inflammation, and regulation of the plasminogen activating system.

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.

Thiazolidinediones inhibit TNFalpha induction of PAI-1 independent of PPARgamma activation.

Increased plasminogen activator inhibitor type 1 (PAI-1) levels are observed in endothelial cells stimulated by tumour necrosis factor alpha (TNFalpha). Thiazolidinediones (TZDs) may inhibit elevated endothelial cell PAI-1 accounting, in part, for the putative atheroprotective effects of TZDs. In an endothelial cell line, Rosiglitazone (RG) and Pioglitazone (PG) inhibited induction of PAI-1 by TNFalpha. The specific peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor, SR-202, failed to modulate this effect. RG also inhibited the effect of TNFalpha on a reporter gene construct harbouring the proximal PAI-1 promoter and PAI-1 mRNA in cells co-transfected with a dominant-negative PPARgamma construct. RG and PG attenuated TNFalpha-mediated induction of trans-acting factor(s) Nur77/Nurr1 and binding of nuclear proteins (NP) to the cis-acting element (NBRE). SR-202 failed to modulate these effects. The observations suggest TZDs inhibit TNFalpha-mediated PAI-1 induction independent of inducible PPARgamma activation and this may involve in the modulation of Nur77/Nurr1 expression and NP binding to the PAI-1 NBRE.

In vivo and in vitro analysis of the human tissue-type plasminogen activator gene promoter in neuroblastomal cell lines: evidence for a functional upstream kappaB element.

Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells.

Control elements between -9.5 and -3.0 kb in the human tissue-type plasminogen activator gene promoter direct spatial and inducible expression to the murine brain.

Tissue-type plasminogen activator (t-PA) participates in the control of synaptic plasticity and memory formation in the central nervous system (CNS). Transgenic mice harbouring either 9.5, 3.0 or 1.4 kb of the human t-PA promoter fused to the LacZ reporter gene were used to assess t-PA promoter-directed expression in vivo. The 9.5 kb t-PA promoter directed expression to the brain, most notably to the dentate gyrus, superior colliculus, hippocampus, thalamus and piriform cortex. Staining was also observed in the retrosplenial and somatosensory cortex. The 3.0 kb t-PA promoter directed generalized and poorly defined expression to the cortex and hippocampus, while the 1.4 kb t-PA promoter directed expression selectively to the medial habenula. Intravenous administration of lipopolysaccharide into mice harbouring the 9.5 kb t-PA promoter resulted in an increase in reporter gene activity in the lateral orbital cortex and thalamus. Results of in vitro transfection experiments of NT2 cells with a series of t-PA promoter deletion constructs confirmed the presence of regulatory elements throughout the 9.5 kb promoter region. Finally, we describe a cis-acting element related to the NFAT recognition site that provides a protein-binding site and which may play a role in the selective expression of the 1.4 t-PA promoter in the medial habenula. These results indicate that elements between -3.0 and -9.5 kb of the t-PA promoter confer constitutive and inducible expression to specific regions of the CNS.

Physiologic concentrations of magnesium and placental apoptosis: prevention by antioxidants.

OBJECTIVE: To identify the role of physiologic magnesium concentrations on the induction of placental apoptosis in vitro and test the anti-apoptotic action of antioxidants. METHODS: Placental tissue was obtained from normal pregnancies after cesarean delivery. Placental explants were incubated with increasing concentrations of extracellular magnesium (range 0-2.0 mM). Placental apoptosis was evaluated by tissue morphology, DNA fragmentation, cytokeratin-18 neoepitope formation, and cleavage of plasminogen activator inhibitor type 2. RESULTS: Physiologic concentrations of extracellular magnesium stimulated placental apoptosis. Magnesium stimulated apoptosis within the physiologic range (0.8-1.2 mM) (n = 6, P <.001) and was associated with cleavage of plasminogen activator inhibitor type 2 and cytokeratin-18 neoepitope formation. These data implicate caspase activation in the transduction of the magnesium-induced apoptotic signal. Therapeutic concentrations of vitamin C, vitamin E, and acetylcysteine (all at 25 microg/mL) inhibited DNA fragmentation and attenuated cleavage of plasminogen activator inhibitor type 2 and cytokeratin-18 neoepitope formation. CONCLUSION: Magnesium-induced placental apoptosis is a potent mechanism of placental degeneration in vitro and may represent an important regulator of placental tissue dynamics in vivo. The ability of antioxidants to prevent magnesium-induced placental apoptosis implicates oxidation-reduction-dependent signaling events in this process. Furthermore, these findings provide a basis for further studies of antioxidants in mitigating the adverse effects of preeclampsia.

Plasminogen activator inhibitor type 2 contains mRNA instability elements within exon 4 of the coding region. Sequence homology to coding region instability determinants in other mRNAs.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that inhibits urokinase. Constitutive and regulated PAI-2 gene expression involves post-transcriptional events, and an AU-rich mRNA instability motif within the 3'-untranslated region of PAI-2 mRNA is required for this process (Maurer, F., Tierney, M., and Medcalf, R. L. (1999) Nucleic Acids Res. 27, 1664-1673). Here we show that instability determinants are present within various exons of the PAI-2 coding region, most notably within exon 4. Deletion of exon 4 from the full-length PAI-2 cDNA results in a doubling in the half-life of PAI-2 mRNA, whereas a 28-nucleotide region within exon 4 contains binding sites for cytoplasmic proteins. Inducible stabilization of PAI-2 mRNA in HT-1080 cells treated with phorbol ester and tumor necrosis factor does not alter the binding of proteins to the exon 4 instability determinant, but resulted in a transient increase in the binding of factors to the AU-rich RNA instability element. Hence, PAI-2 mRNA stability is influenced by elements located within both the coding region and the 3'-untranslated region and that cytoplasmic mRNA binding factors may influence steady state and inducible PAI-2 mRNA expression. Finally a 10-nucleotide region flanking the exon 4 protein-binding site is homologous to instability elements within five other transcripts, suggesting that a common coding region determinant may exist.

Overexpression of a dominant negative CREB protein in HT-1080 cells selectively disrupts plasminogen activator inhibitor type 2 but not tissue-type plasminogen activator gene expression.

The tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 2 (PAI-2) genes are differentially regulated by 12-phorbol 13-myristate acetate (PMA) in HT-1080 fibrosarcoma cells. PMA transcriptionally down-regulates the t-PA gene in HT-1080 cells, while the PAI-2 gene is simultaneously induced by this agonist. The t-PA and PAI-2 gene promoters harbour a cAMP-response element (CRE) which influences the expression of both genes. We have compared the binding activity of nuclear factors that recognise these CRE sites. We show that CREB (CRE binding protein) recognises each CRE and that the degree of constitutive Ser119-phosphorylated t-PA CRE-bound CREB was greater than for PAI-2 CRE bound CREB. Stable transfection of HT-1080 cells with a plasmid containing a CREB that could not be phosphorylated on Ser119 (pCI-CREB(ala119)) did not influence PMA-mediated suppression of t-PA mRNA, but markedly impaired PMA-mediated induction of PAI-2 mRNA. Our results demonstrate that the Ser119 residue of CREB plays a crucial role in PMA-mediated induction of PAI-2 gene expression, whereas PMA-mediated suppression of t-PA in HT-1080 cells requires a different process.

The molecular basis for the aberrant production of plasminogen activator inhibitor type 2 in THP-1 monocytes.

Plasminogen activator inhibitor type 2 (PAI-2) is a urokinase inhibitor that is expressed primarily in monocytes. THP-1 monocytes, however. contain a unique defect in the production of PAI-2 in that the PAI-2 transcript is truncated and the expressed protein inactive (1). Here we describe the basis of this mutation in THP-1 cells. Southern blot analysis of THP-1-derived genomic DNA indicated that there were no obvious deletions in the structure of the PAI-2 gene. However, assessment of the THP-1-derived PAI-2 transcript by RT-PCR indicated that only exons seven and eight of the normal PAI-2 mRNA could be detected. Cloning of the 5' region of the PAI-2 mRNA by 5-'RACE indicated that the PAI-2 cDNA derived from THP-1 cells is approximately 1329 bp long and contains 180 bp of sequence derived from intron 5. followed by sequences corresponding to exons seven and eight of the normal PAI-2 mRNA. The presence of the intron five fragment in endogenous THP-1 derived PAI-2 mRNA was confirmed by Northern blotting. The absence of any wild-type PAI-2 mRNA in these cells suggests that one copy of the PAI-2 allele has been deleted. The remaining allele producing the truncated mRNA appears to have undergone a translocation event and contains a mutation that has disrupted the splicing of the PAI-2 primary transcript.

The novel anti-tumour agent oxamflatin differentially regulates urokinase and plasminogen activator inhibitor type 2 expression and inhibits urokinase-mediated proteolytic activity.

Cell surface, urokinase (u-PA)-mediated, plasminogen activation has recently been recognised as a process integral to extracellular matrix degradation. The primary inhibitor of u-PA activity in the extracellular matrix is plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor. The malignant metastatic phenotype is associated with excessive and uncontrolled, tumour cell-associated, u-PA-mediated, extracellular matrix degradation. Inhibition of the malignant metastatic phenotype via induction of PAI-2 expression and/or inhibition of u-PA expression may represent a novel means via which the metastatic phenotype can be arrested. Agents capable of inducing PAI-2 and/or inhibiting u-PA activity may restrict u-PA-mediated tumour cell proteolysis and facilitate in the development of therapeutic strategies to combat malignant disease. We have identified the hydroxamic acid derivative oxamflatin, previously noted to revert the malignant phenotype in K-ras-transformed NIH-3T3 cells, as capable of upregulating PAI-2 and simultaneously suppressing u-PA expression in two different cell systems. In addition, zymographic analysis indicated that oxamflatin treatment results in a significant reduction in u-PA proteolytic activity in both HT-1080 fibrosarcoma and U-937 histiocytic lymphoma cells. We postulate that oxamflatin represents a novel means by which induction of PAI-2 and concomitant inhibition of u-PA gene and protein expression can be achieved and may be of benefit in inhibiting the malignant metastatic phenotype.

Methylation-dependent silencing of the testis-specific Pdha-2 basal promoter occurs through selective targeting of an activating transcription factor/cAMP-responsive element-binding site.

In this study, we demonstrate that methylation-dependent repression of the Pdha-2 core promoter is mediated regionally through a consensus activating transcription factor/cAMP-responsive element-binding site located between nucleotides -54 and -62 upstream of the major transcriptional start site. Targeting of the CpG dinucleotide within this cis-element significantly disrupts the ability of this basal promoter to activate gene expression in vitro and completely abolishes promoter activity in vivo. DNase I footprinting experiments indicated that availability of the nuclear factor(s) binding this element is limiting in sexually immature mouse testis, and as such, these factors may play an important role in the coordinate activation of early spermatogenic gene expression. Interestingly, CpG dinucleotides associated with the hypersensitive region flanking the activating transcription factor/cAMP-responsive element-binding site appear to confer some conformational structure on the promoter since mutations at these specific CpG dinucleotides result in elevated basal levels of transcription. This raises the possibility of a potential bifunctional role for CpG dinucleotides in either methylation-dependent or -independent processes. Our data support the notion that hypomethylation and transcription factor recruitment are necessary events that precede gene activation at the early stages of spermatogenesis.

An AU-rich sequence in the 3'-UTR of plasminogen activator inhibitor type 2 (PAI-2) mRNA promotes PAI-2 mRNA decay and provides a binding site for nuclear HuR.

The plasminogen activator inhibitor type 2 (PAI-2) gene is regulated by transcriptional and post-transcriptional processes. We have previously shown that insertion of the 3'-untranslated region (3'-UTR) of PAI-2 mRNA into the 3'-UTR of a beta-globin reporter mRNA reduces constitutive beta-globin mRNA expression and that this requires, at least in part, an AU-rich motif. Here we have directly assessed the role of this motif in PAI-2 mRNA stability using both chimeric and non-chimeric reporter systems. We first show that the full-length PAI-2 mRNA is indeed unstable with a half-life of 1 h. Using the c-fos promoter-driven human growth hormone (HGH) mRNA as a reporter, we demonstrate that the 580 nt 3'-UTR of PAI-2 accelerates chimeric HGH mRNA decay in a process which is dependent on the intact AU-rich sequence. Furthermore, disruption of this motif within a constitutively expressed PAI-2 cDNA produces a 2.5- and 2. 7-fold increase in PAI-2 mRNA and protein levels in HT-1080 cells, respectively. RNA electrophoretic mobility shift and supershift assays indicate that this motif provides a specific binding site for cellular proteins that include nuclear HuR. Taken together, these data show that a correlation exists between the binding of HuR to the AU-rich motif in vitro and the destabilizing properties conferred by this sequence in vivo.

Transcriptional regulation of the tissue-type plasminogen-activator gene in human endothelial cells: identification of nuclear factors that recognise functional elements in the tissue-type plasminogen-activator gene promoter.

The gene encoding human tissue-type plasminogen activator (t-PA) is regulated in a cell-type-specific manner. Previous studies in non-endothelial cells have indicated that basal and phorbol ester mediated induction is controlled by a cAMP response element (CRE) referred to as the tPACRE, and an activating protein 2 (AP-2)-like site. The classification of the AP-2-like site was assigned on the basis of its sequence homology, but has been shown in some cell systems to be recognised by promoter-specific transcription factor-1 (Sp-1). Here, we have investigated the transcriptional regulation of the t-PA gene in endothelial cells and addressed the functional roles of the tPACRE and the Sp-1/AP-2-like sites. 5'-RACE experiments indicate that the t-PA gene uses two transcription initiation sites in these cells with the downstream site being preferred. Functional analyses of the t-PA promoter using reporter-gene constructs transfected into C11STH endothelial cells demonstrate that the first 410 bp of the t-PA promoter confers an increase in reporter-gene activity on treatment with 4beta-phorbol 12-myristate 13-acetate (PMA). Mutagenesis of either the tPACRE or the Sp-1/AP-2 site weakens both basal and inducible expression, while disruption of both sites renders the promoter completely unresponsive. Using supershift assays, we identify the predominant tPACRE-binding proteins in nuclear extracts prepared from both C11STH cells and primary umbilical vein endothelial cells (HUVECs) as activating transcription factor 2, CREB (cAMP-responsive-element-binding protein), CREM (cAMP response element modulator) and c-jun. Treatment of cells with PMA results in a selective recruitment of jun-D to the tPACRE, while Sp-1 was identified as the major transcription factor that recognises the AP-2-like site. Based on this data and previous reports, we have reassigned this as a Sp-1-binding site. Finally, the identification of specific endothelial-derived t-PACRE-binding proteins suggests an integral role for these factors in the regulation of t-PA gene expression in human endothelial cells.

Urokinase-mediated transactivation of the plasminogen activator inhibitor type 2 (PAI-2) gene promoter in HT-1080 cells utilises AP-1 binding sites and potentiates phorbol ester-mediated induction of endogenous PAI-2 mRNA.

Urokinase-type plasminogen activator (u-PA) bound to its receptor, u-PAR, initiates signal transduction pathways able to induce expression of the activator protein-1 (AP-1) family member c-fos [1]. Since transcription factors bound to AP-1 recognition sequences within the PAI-2 gene promoter play a role in basal and phorbol ester-mediated induction of PAI-2 gene expression, we hypothesised that u-PA/u-PAR-mediated modulation of AP-1 activity would in turn influence constitutive and inducible PAI-2 gene expression. Treatment of HT-1080 or U-937 cells with high molecular weight u-PA (HMW u-PA) resulted in induction of nuclear proteins binding to a functional AP-1 element in the proximal PAI-2 promoter. This increase in AP-1 activity correlated with a transactivation of the PAI-2 gene promoter in transiently transfected HT-1080 cells. We also demonstrate the u-PA treatment potentiated phorbol ester (PMA)-mediated induction of PAI-2 mRNA, indicating that u-PA binding produces a bone fide response in vivo.

Inhibition of interleukin 1 beta-converting enzyme-mediated apoptosis of mammalian cells by baculovirus IAP.

Apoptosis can be a potent weapon against viral infection and consequently has selected for viruses carrying antiapoptosis genes. Two baculovirus proteins, IAP and p35, can prevent insect cells from dying in response to infection. p35, which interferes with members of the Ced-3 family of cysteine proteases, can also function in mammalian cells. We investigated the ability of IAP from Orgyia pseudotsugata nuclear polyhedrosis virus to prevent death of mammalian cells. IAP was transiently expressed in mammalian cells and its ability to block cell death caused by expression of interleukin-1 beta converting enzyme (ICE), FADD, or the ICE homologues ICH-1 and ICE-Lap3, was investigated. IAP strongly inhibited ICE- and ICH-1-induced cell death but protected only partially against death by overexpression of FADD and not at all against death due to enforced ICE-Lap3 expression. These results demonstrate that a baculoviral IAP protein can functionally interact with conserved components of the apoptosis machinery in mammalian cells.

Molecular mechanisms governing tumor-necrosis-factor-mediated regulation of plasminogen-activator inhibitor type-2 gene expression.

Plasminogen-activator inhibitor type 2 (PAI-2), a serine protease inhibitor involved in the regulation of urokinase-dependent proteolysis, is also implicated in the inhibition of tumor-necrosis-factor-(TNF)-mediated apoptosis. The PAI-2 gene is one of the most TNF-responsive genes known and is also highly induced by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the phosphatase inhibitor, okadaic acid, in both HT-1080 fibrosarcoma and U-937 histiocytic cells. We sought to identify and characterize regulatory cis-acting DNA elements and trans-acting factors which mediate basal and inducible PAI-2 gene transcription. A series of promoter deletion mutants (nucleotides -1859 to -91) fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into HT-1080 cells. Two repressor regions were identified; one distally between positions -1859 and -1100, and one proximally between positions -259 and -219. Cells transfected with constructs harboring more than 259 bp promoter sequence produced a 10-15-fold increase in CAT activity when treated with PMA or okadaic acid, but produced only a minimal (2.5-fold) increase in response to TNF. Removal of the proximal repressor by deletion to position -219, or by internal deletion from the -1100 PAI-2 CAT construct, resulted in a selective increase in TNF responsiveness, suggesting that induction of PAI-2 gene transcription by TNF is associated with derepression. Detailed analysis of the proximal repressor utilizing the electrophoretic mobility shift assay (EMSA), identified two novel and distinct protein-binding sites (A and B). Site A is located within the 40-bp proximal repressor while site B is situated immediately adjacent to the 3' boundary. Treatment of cells with PMA or okadaic acid produced no change in the binding activity of proteins recognising sites A or B. However, treatment of cells with TNF results in a profound selective reduction in site-B-binding activity, suggesting that this site plays a significant role in TNF-mediated regulation of PAI-2 gene expression. Our findings suggest that TNF-mediated induction of PAI-2 gene expression involves derepression and is associated with cis-acting and trans-acting factors located within and adjacent to the proximal repressor region.