Transcription factor FOXP3: A repressor of the TFPI gene?

Single nucleotide polymorphisms (SNPs) may play an important role in the risk of certain diseases. We have previously shown that the -287T/C SNP of the tissue factor pathway inhibitor (TFPI) gene promoter region exerts differential impact on TFPI mRNA expression; the C allele being associated with higher TFPI expression, which in turn is associated with reduced risk of thrombosis. In the present study, we aimed to reveal the underlying molecular mechanisms using human embryonic kidney 293 (HEK293) and Michigan Cancer Foundation-7 (MCF7) cells that both express TFPI. Transfecting the cells with luciferase reporter gene constructs containing the TFPI promoter with either the T or the C allele of -287T/C resulted in increased luciferase activity with the C allele relative to the T allele. Three potential candidate transcription factors for binding to the two -287 alleles were predicted using the ALGGEN PROMO software, and results from electrophoretic mobility shift assays indicated that forkhead box protein 3 (FOXP3), initially identified as a functional marker of T regulator cells, bound more specifically to the T allele compared with the C allele. By chromatin immunoprecipitation assays analysis it was confirmed that FOXP3 was able to bind to the DNA region that contains the SNP. Knockdown or overexpression of FOXP3 resulted in increased or decreased TFPI levels, respectively, in both cell types. In conclusion, this study indicates that FOXP3 most likely is involved in the increased levels of TFPI observed with the -287C allele and also that FOXP3 might be a repressor for TFPI expression.

Factor VII deficiency: Unveiling the cellular and molecular mechanisms underlying three model alterations of the enzyme catalytic domain.

Activated factor (F) VII is a vitamin K-dependent glycoprotein that initiates blood coagulation upon interaction with tissue factor. FVII deficiency is the most common of the rare congenital bleeding disorders. While the mutational pattern has been extensively characterized, the pathogenic molecular mechanisms of mutations, particularly at the intracellular level, have been poorly defined. Here, we aimed at elucidating the mechanisms underlying altered FVII biosynthesis in the presence of three mutation types in the catalytic domain: a missense change, a microdeletion and a frameshift/elongation, associated with severe or moderate to severe phenotypes. Using CHO-K1 cells transiently transfected with expression vectors containing the wild-type FVII cDNA (FVIIwt) or harboring the p.I289del, p.G420V or p.A354V-p.P464Hfs mutations, we found that the secretion of the FVII mutants was severely decreased compared to FVIIwt. The synthesis rate of the mutants was slower than the FVIIwt and delayed, and no degradation of the FVII mutants by proteasomes, lysosomes or cysteine proteases was observed. Confocal immunofluorescence microscopy studies showed that FVII variants were localized into the endoplasmic reticulum (ER) but were not detectable within the Golgi apparatus. These findings suggested that a common pathogenic mechanism, possibly a defective folding of the mutant proteins, was triggered by the FVII mutations. The misfolded state led to impaired trafficking of these proteins causing ER retention, which would explain the low to very low FVII plasma levels observed in patients carrying these mutations.

Normalization of disrupted clock gene expression in males with tetraplegia: a crossover randomized placebo-controlled trial of melatonin supplementation.

STUDY DESIGN: Crossover double blind, randomized placebo-controlled trial. OBJECTIVES: Circadian oscillators are located both in the brain and in peripheral organs. Melatonin, the main brain-derived hormone governing circadian variations, is highly associated with daylight patterns. However, in subjects with tetraplegia the melatonin levels are blunted. Here we studied peripheral oscillators in peripheral blood mononuclear cells (PBMCs) in males with tetraplegia by examining how exogenous melatonin may influence the expression of clock gene mRNAs. SETTING: Sunnaas Rehabilitation Hospital, Nesoddtangen, Norway. METHODS: Six males with tetraplegia received 2 mg of melatonin or placebo 4 days before the study period. We also included six able-bodied men sleeping or kept awake during the night. Plasma samples were collected four times during a 24-h period. The mRNA expression levels of the clock genes PER1, PER2, BMAL1, and REV-ERBα were quantified in PBMCs using quantitative RT-PCR. RESULTS: The mRNA expression levels of PER-1 and -2 and REV-ERBα were increased at 04:00 h compared with the able-bodied controls (p < 0.05). Melatonin supplementation changed mRNA peak-time toward the time of supplementation. CONCLUSIONS: Several peripheral clock genes displayed distorted expression levels in tetraplegia. Supplementation with melatonin changed the mRNA expression levels of these genes toward those observed among able-bodied. SPONSORSHIP: Financial support was provided from the Throne Holst Foundation, Sunnaas Rehabilitation hospital and the University of Ferrara (FAR2016).

EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells.

Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders.

Tissue factor pathway inhibitor attenuates ER stress-induced inflammation in human M2-polarized macrophages.

Endoplasmic reticulum (ER) stress has been shown to play a key role during the initiation and clinical progression of the cardiovascular diseases, such as atherosclerosis. We have recently shown that expression of tissue factor pathway inhibitor (TFPI) in human monocyte-derived macrophages (MDMs) was induced by cholesterol crystals (CC). In the present study we aimed to determine the role of TFPI under ER stress conditions using human MDMs. qRT-PCR and immunohistochemistry analysis were performed to determine the presence of the ER stress marker CCAAT/enhancer binding protein homologous protein (CHOP) and TFPI in human carotid plaque material and also in human MDMs polarized into pro-inflammatory M1 or anti-inflammatory M2 populations. CHOP mRNA levels were upregulated in the plaques compared to healthy vessels, and CHOP protein was localized in the same area as TFPI in the plaques. Both CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype, and the ER stress inhibitor 4-phenylbutyric acid (PBA) reversed this effect. Furthermore, CC treatment increased the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, which for TNF-α and IL-8 was inhibited by PBA, and reduced the levels of the anti-inflammatory cytokine IL-10 in M2-polarized macrophages. Knockdown of TFPI prior to CC treatment exacerbated TNF-α and IL-6 levels, but reduced IL-8 and IL-10 levels. Our results show that CC induce TFPI and cytokine expression in M2-polarized macrophages through activation of the ER stress pathway and that TFPI has a protective effect against TNF-α and IL-6 mediated inflammation. These mechanisms may have implications for the pathogenesis of atherosclerosis.

Increased coagulation activity and genetic polymorphisms in the F5, F10 and EPCR genes are associated with breast cancer: a case-control study.

BACKGROUND: The procoagulant state in cancer increases the thrombotic risk, but also supports tumor progression. To investigate the molecular mechanisms controlling cancer and hemostasis, we conducted a case-control study of genotypic and phenotypic variables of the tissue factor (TF) pathway of coagulation in breast cancer. METHODS: 366 breast cancer patients and 307 controls were genotyped for SNPs (n = 41) in the F2, F3 (TF), F5, F7, F10, TFPI and EPCR genes, and assayed for plasma coagulation markers (thrombin generation, activated protein C (APC) resistance, D-dimer, antithrombin, protein C, protein S, and TF pathway inhibitor (TFPI)). Associations with breast cancer were evaluated using logistic regression to obtain odds ratios (ORs) and 95% confidence intervals (CIs), or the chi-square test. RESULTS: Four SNPs in F5 (rs12120605, rs6427202, rs9332542 and rs6427199), one in F10 (rs3093261), and one in EPCR (rs2069948) were associated with breast cancer. EPCR rs2069948 was associated with estrogen receptor (ER) and progesterone receptor (PR) positivity, while the SNPs in F5 appeared to follow hormone receptor negative and triple negative patients. The prothrombotic polymorphisms factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) were not associated with breast cancer. High APC resistance was associated with breast cancer in both factor V Leiden non-carriers (OR 6.5, 95% CI 4.1-10.4) and carriers (OR 38.3, 95% CI 6.2-236.6). The thrombin parameters short lag times (OR 5.8, 95% CI 3.7-9.2), short times to peak thrombin (OR 7.1, 95% CI 4.4-11.3), and high thrombin peak (OR 6.1, 95% CI 3.9-9.5) predicted presence of breast cancer, and high D-dimer also associated with breast cancer (OR 2.0, 95% CI 1.3-3.3). Among the coagulation inhibitors, low levels of antithrombin associated with breast cancer (OR 5.7, 95% CI 3.6-9.0). The increased coagulability was not explained by the breast cancer associated SNPs, and was unaffected by ER, PR and triple negative status. CONCLUSIONS: A procoagulant phenotype was found in the breast cancer patients. Novel associations with SNPs in F5, F10 and EPCR to breast cancer susceptibility were demonstrated, and the SNPs in F5 were confined to hormone receptor negative and triple negative patients. The study supports the importance of developing new therapeutic strategies targeting coagulation processes in cancer.

Hypoxia influences stem cell-like properties in multidrug resistant K562 leukemic cells.

OBJECTIVES: The present study investigates the potential role of hypoxia in maintaining stem cell-like properties and therapeutic resistance in K562 leukemic cell. METHODS: Western blot, flow cytometry and cell viability assays were used to investigate the effects of hypoxia (1% O2) on cell proliferation, drug resistance and expression of the hypoxia inducible factor-2α (HIF-2α), the octamer-binding transcription factor 4 (Oct4), CD133, CD34 and the ATP-binding cassette sub-family G member 2 (ABCG2) as well as Smad2 phosphorylation in the drug resistant cell line K562/DOX and its parental cell line. RESULTS: Hypoxia induced growth inhibition and significantly upregulated HIF-2α, CD133, Oct4, CD34 and ABCG2 expression in the wild type K562 cells (p<0.05). The IC50 of doxorubicin was also enhanced about 2.5-fold in hypoxia. In contrast, the K562/DOX cells, which showed significantly higher ABCG2 expression and IC50 for various drugs, no significant difference in cell proliferation was observed between hypoxia and normoxia. The hypoxia-induced upregulation of HIF-2α, CD133, Oct4, CD34 and ABCG2 expression was significantly lower than in the wild type cells (p<0.05). Moreover, hypoxia induced the phosphorylation of Smad2 and additional treatment with SD-208, an inhibitor of the TGF-ß receptor I kinase, resulted in a dose-dependent downregulation of CD133 and Oct4 in the K562/DOX cells. CONCLUSIONS: Hypoxia plays an important role in enhancing the stem cell-like properties and to induce multidrug resistance of leukemia cells. The activation of the TGF-ß/Smad2 signaling pathway may be involved in the regulation of this pathophysiological process.

Candidate gene polymorphisms and the risk for pregnancy-related venous thrombosis.

Venous thrombosis (VT) is one of the leading causes of maternal death in the western world, but the genetic causes of pregnancy-related VT are insufficiently understood. The aim of this study was to investigate the association between common genetic variations in candidate genes and pregnancy-related VT. We undertook a hospital based case-control study of women with VT during pregnancy or puerperium; controls were women giving birth without having VT. Single nucleotide polymorphisms (SNPs) were selected in 49 pre-specified candidate genes involved in coagulation, inflammation, and hormonal metabolism in 313 cases and 353 controls. We found new associations between SNPs and total pregnancy-related VT in the genes encoding coagulation factors V and VIII, and p-selectin. Additional new associations between SNPs and antenatal VT were found in the genes encoding the epidermal growth factor receptor, the pregnane X receptor, and protein S. Of 21 SNPs previously associated with thrombotic disease, rs2289252 in F11 and rs3917643 in F3 were associated with pregnancy-related VT, while rs4524 in F5 was associated with antenatal VT.

Overexpression of tissue factor pathway inhibitor in CHO-K1 cells results in increased activation of NF-κB and apoptosis mediated by a caspase-3 independent pathway.

There is now circumstantial evidence that tissue factor pathway inhibitor (TFPI) is not only a major anticoagulant, but also has proapoptotic properties. The current study was designed to address the role of TFPI on signalling pathways and apoptosis. The non-TFPI expressing cell line CHO-K1 was stably transfected with pcDNA3.1/V5-His-TOPO-TFPI and control cells were established by transfecting the CHO-K1 cells with pcDNA3.1/V5-His-TOPO. Sodium butyrate (NaBut) has been shown to induce the expression of recombinant proteins. Here we have used NaBut to increase the expression of TFPI as assessed by qRT-PCR and ELISA. Compared to the control cells, TFPI induced apoptosis in a concentration dependent manner as measured by a cell death detection assay. Independent of caspase-3 activation an increased cleavage of PARP was detected in the TFPI expressing cells. This was accompanied by downregulation of Bcl-XL, elevated levels of Bax, and increased translocation of the apoptosis initiating factor. Increased DNA binding activity of NF-κB was revealed by electrophoretic mobility shift assay when the TFPI level was elevated by NaBut together with an increased translocation of the NF-κB subunit p65. The results indicate that TFPI affected the apoptotic activity through a process independent of caspase-3, and was also able to increase the activation of the NF- κB pathway.

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility.

BACKGROUND: Increased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231). METHODS: Stable cell lines with TFPI (both α and ß) and only TFPIß downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion. RESULTS: Downregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIß was downregulated, revealing a novel function of this isoform in cancer metastasis. CONCLUSIONS: Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.

Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport.

BACKGROUND: Activated protein C (PC) is a serine protease that regulates blood coagulation by inactivating coagulation factors Va and VIIIa. PC deficiency is an autosomally inherited disorder associated with a high risk of recurrent venous thrombosis. The aim of the study was to explore the mechanisms responsible for severe PC deficiency in a patient with the protein C A267T mutation by in-vitro expression studies. RESULTS: Huh7 and CHO-K1 cells were transiently transfected with expression vectors containing wild-type (WT PC) and mutated PC (A267T PC) cDNAs. PC mRNA levels were assessed by qRT-PCR and the PC protein levels were measured by ELISA. The mRNA levels of WT PC and A267T PC were similar, while the intracellular protein level of A267T PC was moderately decreased compared to WT PC. The secretion of A267T PC into the medium was severely impaired. No differences in molecular weights were observed between WT and A267T PC before and after treatment with endo-ß-N-acetylglucosaminidase. Proteasomal and lysosomal degradations were examined using lactacystin and bafilomycin, respectively, and revealed that A267T PC was slightly more susceptible for proteasomal degradation than WT PC. Intracellular co-localization analysis indicated that A267T PC was mainly located in the endoplasmic reticulum (ER), whereas WT PC was observed in both ER and Golgi. CONCLUSIONS: In contrast to what has been reported for other PC mutants, intracellular degradation of A267T PC was not the main/dominant mechanism underlying the reduced intracellular and secretion levels of PC. Our results indicate that the A267T mutation most likely caused misfolding of PC, which might lead to increased retention of the mutated PC in ER.

Functional characterization of polymorphisms in the human TFPI gene.

Tissue factor pathway inhibitor (TFPI) is the primary physiological inhibitor of tissue factor (TF) induced coagulation. Low plasma TFPI levels have been shown to be associated with increased risk of arterial and venous thrombosis. Several clinical studies have reported that single nucleotide polymorphisms (SNPs) in the regulatory regions of the gene, such as the -287T/C, the -399C/T, and the -33T/C SNPs, may affect plasma TFPI levels. However, molecular studies investigating the functionality of the polymorphisms are lacking. In this study, we found that the -287C and -399T alleles affected the activity of the promoter using a reporter gene system. This was also the case for the -33T/C polymorphism. An association regarding the transcriptional activity of the reporter gene was detected between the -287C allele and the -33T/C polymorphism. Analysis of the polymorphic sites with electrophoretic mobility shift assay (EMSA) showed that all three polymorphisms potentially alter DNA-protein interactions. Based on these findings, we speculate that the -287C and the -33C alleles can be associated with lowered risk of thrombosis.

Overexpression of both TFPIα and TFPIß induces apoptosis and expression of genes involved in the death receptor pathway in breast cancer cells.

Thrombosis is a major complication and an important cause of death in cancer patients. Tumor cells may trigger coagulation and induce a prothrombotic phenotype, which in return may enhance angiogenesis, tumor growth, and metastasis. Tissue factor pathway inhibitor (TFPI) has been reported to reduce tumor growth and metastasis in vivo and to induce apoptosis and inhibit proliferation in normal cells in vitro. However, no effect has so far been observed in cancer cells. We therefore aimed to characterize the functional effects of ectopic overexpression and endogenous downregulation of TFPI in cancer cells, and to elucidate possible mechanisms involved. The tumor derived breast cancer cells SK-BR-3 and Sum102 were used to construct stable cell lines overexpressing TFPIα and TFPIß, and with TFPI knocked down, respectively. Effects of altered TFPI expression were evaluated by measuring apoptosis and proliferation of the cells, and gene expressions were analyzed using PCR arrays. Increased DNA fragmentation and Caspase 3 activity was observed in SK-BR-3 cells overexpressing TFPIα and TFPIß, while a decrease in apoptosis was seen in Sum102 cells with TFPI expression knocked down. An increase and reduction in expression of pro- and anti-apoptotic genes, respectively, were seen in TFPI overexpressing cells, and the majority of the upregulated genes encoded proteins involved in the death receptor pathway, among them the death receptor ligand TNF-α. In conclusion, TFPIα and TFPIß induced apoptosis in breast cancer cells and increased expression of apoptotic genes indicating a possible involvement of the death receptor pathway.

Identification of differentially expressed genes associated with osteochondrosis in standardbred horses using RNA arbitrarily primed PCR.

The aim of this study was to investigate genes for differential expression in cartilage of foals predisposed to osteochondrosis (OC). Tissue was sampled from the cranial part of the distal intermediate ridge of the tibia in the tarso-crural joint. Foals were considered predisposed to OC when parents had OC at the distal intermediate ridge of the tibia. RNA was isolated and subjected to arbitrarily primed PCR (RAP-PCR) followed by fingerprinting to screen for differentially expressed genes. By verification of results from the RAP-PCR fingerprint screening using real-time RT-PCR, we identified two genes not previously correlated with OC as differentially expressed. The two genes, which were identical to TLK2 and an equine EST, are good targets for future research on OC.

Indirect regulation of TFPI-2 expression by miR-494 in breast cancer cells.

TFPI-2 has been shown to be involved in breast cancer pathogenesis by inhibiting extracellular matrix degradation, and low levels are associated with disease progression. As microRNA-494 (miR-494) protects against breast cancer progression, we investigated whether miR-494 is involved in the regulation of TFPI-2 in MCF-7 breast cancer cells. TFPI-2 mRNA and protein levels increased after transfection with miR-494 mimic, and TFPI-2 mRNA and miR-494 levels correlated positively in tumors from breast cancer patients. No specific binding sites for miR-494 in the 3'-untranslated region (UTR) of TFPI2 were identified; however, miR-494 was predicted in silico to bind 3'-UTR of the transcription factors AHR and ELF-1, which have potential binding sites in the TFPI2 promoter. ELF-1 mRNA was downregulated whereas AHR mRNA levels were upregulated after transfection with miR-494 mimic. Knockdown of ELF-1 and AHR increased and reduced TFPI-2 mRNA levels, respectively. Increased luciferase activity was seen when TFPI-2 promoter constructs containing the potential AHR or ELF-1 binding sites were co-transfected with miR-494 mimic. In conclusion, TFPI-2 mRNA levels were upregulated by miR-494 in MCF-7 breast cancer cells most likely by an indirect association where miR-494 targeted the transcription factors AHR and ELF-1. This association was supported in a breast cancer cohort.

Expression of the V264M TFPI mutant in endothelial cell cultures may involve mRNA stability.

INTRODUCTION: Tissue factor (TF) pathway inhibitor (TFPI) is the endogenous inhibitor regulating TF-induced blood coagulation. Several polymorphisms have been identified in the TFPI gene and some of them have been correlated with variations in plasma TFPI levels. The aim of the present study was to characterize the TFPI(V264M) mutant in comparison with the wild type protein (TFPI(WT)). MATERIALS AND METHODS: We have overexpressed the TFPI(V264M) mutant and TFPI(WT) in human coronary artery endothelial cells and compared the expression and activity levels of the mutated protein relative to the TFPI(WT). The protein levels were determined by ELISA, the inhibitory activity of the proteins was assessed with a chromogenic substrate assay. The mRNA level of the two TFPI variants was determined using real time RT-PCR. MFOLD was used to predict mRNA secondary structure. RESULTS AND CONCLUSIONS: TFPI(V264M) displayed increased protein levels and activity compared to TFPI(WT) accompanied by an increase in mRNA levels of TFPI(V264M) due to prolonged stability of TFPI(V264M) mRNA. The specific activity of the TFPI(V264M) was similar to TFPI(WT), indicating that the mutation does not affect the enzymatic function of the protein.

The effect of the chemical chaperone 4-phenylbutyrate on secretion and activity of the p.Q160R missense variant of coagulation factor FVII.

BACKGROUND: Congenital coagulation factor (F) VII deficiency is a rare bleeding disorder caused by mutations in the F7 gene. The missense factor FVII variant p.Q160R is the disease-causing mutation in all Norwegian FVII deficient patients and results in reduced biological activity and antigen levels of FVII in patient plasma. Previous in vitro studies on this variant demonstrated impaired intracellular trafficking and reduced secretion, possibly due to protein misfolding. The aim of the study was therefore to assess the impact of chemical chaperones on cellular processing and secretion of this variant using a cell model based on overexpression of the recombinant protein. RESULTS: Through screening of compounds, we identified 4-phenylbutyrate (4-PBA) to increase the secretion of recombinant (r) FVII-160R by ~ 2.5-fold. Additionally, treatment with 4-PBA resulted in a modest increase in specific biological activity. Intracellular localization studies revealed that upon treatment with 4-PBA, rFVII-160R was secreted through Golgi and Golgi reassembly-stacking protein (GRASP)-structures. CONCLUSIONS: The present study demonstrates that the chemical chaperone 4-PBA, restores intracellular trafficking and increases the secretion of a missense FVII variant with functional properties in the extrinsic coagulation pathway.

Identification of differentially expressed genes in ileal Peyer's patch of scrapie-infected sheep using RNA arbitrarily primed PCR.

BACKGROUND: In scrapie and prion diseases, the knowledge concerning genes involved in host response during the early infection period in the lymphoid tissues, still remains limited. In the present study, we have examined differential gene expression in ileal Peyer's patches and in laser microdissected follicles of sheep infected with scrapie. METHODS: Ileal Peyer's patches and laser microdissected follicles were of scrapie and control lambs with susceptible genotypes for classical scrapie. Potential regulated genes were found using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and fingerprinting. The differentially expressed genes were confirmed using real-time RT-PCR. RESULTS: The expression of three genes (MAPRE3, LOC729073 and DNAJC3), were found to be significantly altered in scrapie infected lambs (P < 0.05). CONCLUSION: The three genes have not previously been associated with prion diseases and are interesting as they may reflect biological processes involved in the molecular pathogenesis of prion diseases.

Activation of Endoplasmic Reticulum Stress and Unfolded Protein Response in Congenital Factor VII Deficiency.

Congenital factor (F) VII deficiency is a bleeding disorder caused by a heterogeneous pattern of mutations in the F7 gene. Protein misfolding due to mutations is a strong candidate mechanism to produce the highly represented type I FVII deficiency forms, characterized by a concomitant deficiency of FVII antigen and activity. Misfolded proteins can accumulate within the endoplasmic reticulum (ER) causing ER stress with subsequent activation of the unfolded protein response (UPR). So far, there are limited data on this important issue in FVII deficiency. In this study, we chose as candidate FVII model mutations, the p.Q160R, p.I289del and p.A354V-p.P464Hfs, which are all associated with severe to moderate type I FVII deficiency. In vitro expression of the recombinant (r) mutants rFVII-160R, rFVII-289del or rFVII-354V-464Hfs, which are characterized by either amino acid substitution, deletion, or by an extended carboxyl terminus, demonstrated inefficient secretion of the mutant proteins, probably caused by intracellular retention and association with ER chaperones. Both ER stress and UPR were activated following expression of all FVII mutants, with the highest response for rFVII-289del and rFVII-354V-464Hfs. These data unravel new knowledge on pathogenic mechanisms leading to FVII deficiency, and support the investigation of pharmaceutical modulators of ER stress and UPR as therapeutic agents.

Estrogen induced expression of tissue factor pathway inhibitor-2 in MCF7 cells involves lysine-specific demethylase 1.

Hormone-sensitive cancers can be influenced by estrogens, a process usually mediated through the estrogen receptor (ER). Tissue factor pathway inhibitor type 2 (TFPI-2) is a Kunitz-type serine protease inhibitor involved in regulating the extracellular matrix. The present study demonstrates that the expression of TFPI-2 can be induced by estrogens. Breast cancer data from GOBO displayed increased levels of TFPI-2 and increased survival in patients with ERα+ tumors. Treatment of MCF7 cells (ERα+) with 17ß-estradiol (E2) or 17α-ethinyl estradiol (EE2) increased TFPI-2 mRNA and protein levels. This effect was mitigated with fulvestrant and by knocking down ERα, indicating that estrogen mediated TFPI-2 induction was through ERα. Upon knock down of DNA cytosine-5 methyltransferase 1 (DNMT1) or lysine-specific demethylase 1 (LSD1) in MCF7 cells, reduced effect of E2 on TFPI-2 mRNA levels was observed. Our data thus suggest that estrogen induced TFPI-2 expression in MCF7 cells is mediated by ERα and also by the action of LSD1.