Identification of genomic loci regulating platelet plasminogen activator inhibitor-1 in mice.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1, Serpine1) is an important circulating fibrinolysis inhibitor. PAI-1 exists in 2 pools, packaged within platelet α-granules and freely circulating in plasma. Elevated plasma PAI-1 levels are associated with cardiovascular disease. However, little is known about the regulation of platelet PAI-1 (pPAI-1). OBJECTIVES: We investigated the genetic control of pPAI-1 levels in mice and humans. METHODS: We measured pPAI-1 antigen levels via enzyme-linked immunosorbent assay in platelets isolated from 10 inbred mouse strains, including LEWES/EiJ (LEWES) and C57BL/6J (B6). LEWES and B6 were crossed to produce the F1 generation, B6LEWESF1. B6LEWESF1 mice were intercrossed to produce B6LEWESF2 mice. These mice were subjected to genome-wide genetic marker genotyping followed by quantitative trait locus analysis to identify pPAI-1 regulatory loci. RESULTS: We identified differences in pPAI-1 between several laboratory strains, with LEWES having pPAI-1 levels more than 10-fold higher than those in B6. Quantitative trait locus analysis of B6LEWESF2 offspring identified a major pPAI-1 regulatory locus on chromosome 5 from 136.1 to 137.6 Mb (logarithm of the odds score, 16.2). Significant pPAI-1 modifier loci on chromosomes 6 and 13 were also identified. CONCLUSION: Identification of pPAI-1 genomic regulatory elements provides insights into platelet/megakaryocyte-specific and cell type-specific gene expression. This information can be used to design more precise therapeutic targets for diseases where PAI-1 plays a role.

Platelet tissue factor pathway inhibitor-α dampens cardiac thrombosis and associated fibrosis in mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of events initiating the blood coagulation pathway. Tfpi-/- mice die during embryonic development. The absence of protease-activated receptor (PAR) 4, the major thrombin receptor on mouse platelets, rescues Tfpi-/-mice to adulthood. Among the 3 TFPI isoforms in mice, TFPIα is the only isoform within platelets (pltTFPIα) and the only isoform that inhibits prothrombinase, the enzymatic complex that converts prothrombin to thrombin. OBJECTIVES: To determine biological functions of pltTFPIα. METHODS: Tfpi-/-/Par4-/- mice were irradiated and transplanted with bone marrow from mice lacking or containing pltTFPIα. Thus, PAR4 expression was restored in the recipient mice, which differed selectively by the presence or absence of pltTFPIα and lacked other forms of TFPI. RESULTS: Recipient mice lacking pltTFPIα had reduced survival over the 200-day posttransplant period. Necropsy revealed radiation injury associated with large intraventricular platelet-rich thrombi, whereas other organs were not affected. Thrombi were associated with fibrotic presentations, including increased collagen deposition, periostin-positive activated fibroblasts, myofibroblasts, and macrophage infiltrates. Recipient mice containing pltTFPIα showed evidence of radiation injury but lacked heart pathology. CONCLUSIONS: Tfpi-/-/Par4-/- mice develop severe cardiac fibrosis following irradiation and transplantation with bone marrow lacking pltTFPIα. This pathology is markedly reduced when the mice are transplanted with bone marrow containing pltTFPIα. Thus, in this model system pltTFPIα has an important physiological role in dampening pathological responses mediated by activated platelets within the heart tissue.

Genetic analysis of human RNA binding motif protein 48 (RBM48) reveals an essential role in U12-type intron splicing.

U12-type or minor introns are found in most multicellular eukaryotes and constitute ∼0.5% of all introns in species with a minor spliceosome. Although the biological significance for the evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome, predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA binding motif protein 48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5' end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron-containing genes have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of minor intron-containing genes disrupted in both species. Mutations in the majority of these orthologous minor intron-containing genes have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.

The contribution of TFPIα to the hemostatic response to injury in mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an essential regulator of coagulation, limiting thrombin generation and preventing thrombosis. In humans and mice, TFPIα is the sole isoform present in platelets. OBJECTIVE: Here, we asked whether TFPIα, because of its release from platelets at sites of injury, has a unique role in limiting the hemostatic response. METHODS: TFPIα-mutant (TfpiΔα/Δα ) mice were generated by introducing a stop codon in the C-terminus. Platelet accumulation, platelet activation, and fibrin accumulation were measured following penetrating injuries in the jugular vein and cremaster muscle arterioles, and imaged by fluorescence and scanning electron microscopy. Time to bleeding cessation was recorded in the jugular vein studies. RESULTS: TfpiΔα/Δα mice were viable and fertile. Plasma TFPI levels were normal in the TfpiΔα/Δα mice, no TFPI protein or activity was present in their platelets and thrombin-antithrombin complex levels were indistinguishable from Tfpi+/+ littermates. There was a small, but statistically significant reduction in the time to bleeding cessation following jugular vein puncture injury in the TfpiΔα/Δα mice, but no measurable changes in platelet or fibrin accumulation or in hemostatic plug architecture following injury of the micro- or macrovasculature. CONCLUSION: Loss of TFPIα expression does not produce a global prothrombotic state in mice. Platelet TFPIα is expected to be released or displayed in a focal manner at the site of injury, potentially accumulating to high concentrations in the narrow gaps between platelets. If so, the data from the vascular injury models studied here indicate this is not essential for a normal hemostatic response in mice.

Intrauterine lethality in Tfpi gene disrupted mice is differentially suppressed during mid- and late-gestation by platelet TFPIα overexpression.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein required for murine embryonic development. Intrauterine lethality of Tfpi-/- mice occurs at mid- and late gestation, the latter of which is associated with severe cerebrovascular defects. Megakaryocytes produce only the TFPIα isoform, which is stored within platelets and released upon activation. OBJECTIVES: To examine biological activities of platelet TFPIα (pTFPIα) by characterizing effects of pTFPIα overexpression in Tfpi-/- mice. METHODS: Transgenic mice overexpressing pTFPIα were generated and crossed onto the Tfpi-/- background. Genetic and histological analyses of embryos were performed to investigate the function of pTFPIα during embryogenesis. RESULTS: The transgene (Tg) increased pTFPIα 4- to 5-fold without altering plasma TFPI in adult Tfpi+/+ and Tfpi+/- mice but did not rescue Tfpi-/- mice to wean. Analyses of the impact of pTFPIα overexpression on Tfpi-/- survival, however, were complicated by linkage between the Tg integration site and the endogenous Tfpi locus on chromosome 2. Strain-specific genetic interactions also modulated Tfpi-/- embryonic survival. After accounting for these underlying genetic factors, pTFPIα overexpression completely suppressed mid-gestational lethality of Tfpi-/- embryos but had no effect on development of cerebrovascular defects during late gestation resulting in their lack of survival to wean. CONCLUSIONS: pTFPIα overexpression rescued Tfpi-/- embryos from mid-gestational but not late gestational lethality. The prevalence of underlying genetic factors complicating analyses within our study illustrates the importance of meticulously characterizing transgenic mouse models to avoid spurious interpretation of results.

Tissue factor pathway inhibitor is required for cerebrovascular development in mice.

Tissue factor pathway inhibitor (TFPI) inhibits proteases in the blood coagulation cascade that lead to the production of thrombin, including prothrombinase (factor Xa [FXa]/FVa), the catalytic complex that directly generates thrombin. Thus, TFPI and FV are directly linked in regulating the procoagulant response. Studies using knockout mice indicate that TFPI and FV are necessary for embryogenesis, but their contributions to vascular development are unclear. We performed extensive histological analyses of Tfpi-/- and Tfpi-/-F5-/- mouse embryos to investigate the importance of the interplay between TFPI and FV in regulating hemostasis and vascular development during embryogenesis. We observed normal tissue development throughout Tfpi-/- embryos, except in the central nervous system (CNS). The CNS displayed stunted brain growth, delayed development of the meninges, and severe vascular pathology characterized by the formation of glomeruloid bodies surrounding areas of cellular death, fibrin deposition, and hemorrhage. Removing FV from Tfpi-/- embryos completely ameliorated their brain pathology, suggesting that TFPI dampens FV-dependent procoagulant activity in a manner that modulates cerebrovascular development. Thus, we have identified a previously unrecognized role for TFPI activity within the CNS. This TFPI activity likely diminishes an effect of excess thrombin activity on signaling pathways that control cerebral vascular development.

Deficiency of plasminogen activator inhibitor-2 results in accelerated tumor growth.

BACKGROUND: Upregulation of the plasminogen activation system, including urokinase plasminogen activator (uPA), has been observed in many malignancies, suggesting that co-opting the PA system is a common method by which tumor cells accomplish extracellular matrix proteolysis. PAI-2, a serine protease inhibitor, produced from the SERPINB2 gene, inhibits circulating and extracellular matrix-tethered uPA. Decreased SERPINB2 expression has been associated with increased tumor invasiveness and metastasis for several types of cancer. PAI-2 deficiency has not been reported in humans and PAI-2-deficient (SerpinB2-/- ) mice exhibit no apparent abnormalities. OBJECTIVES: We investigated the role of PAI-2 deficiency on tumor growth and metastasis. METHODS: To explore the long-term impact of PAI-2 deficiency, a cohort of SerpinB2-/- mice were aged to >18 months, with spontaneous malignancies observed in 4/9 animals, all of apparently vascular origin. To further investigate the role of PAI-2 deficiency in malignancy, SerpinB2-/- and wild-type control mice were injected with either B16 melanoma or Lewis lung carcinoma tumor cells, with markedly accelerated tumor growth observed in SerpinB2-/- mice for both cell lines. To determine the relative contributions of PAI-2 from hematopoietic or nonhematopoietically derived sources, bone marrow transplants between wild-type C57BL/6J and SerpinB2-/- mice were performed. RESULTS AND CONCLUSIONS: Our results suggest that PAI-2 deficiency increases susceptibility to spontaneous tumorigenesis in the mouse, and demonstrate that SerpinB2 expression derived from a nonhematopoietic compartment is a key host factor in the regulation of tumor growth in both the B16 melanoma and Lewis lung carcinoma models.

RNA Binding Motif Protein 48 Is Required for U12 Splicing and Maize Endosperm Differentiation.

The last eukaryotic common ancestor had two classes of introns that are still found in most eukaryotic lineages. Common U2-type and rare U12-type introns are spliced by the major and minor spliceosomes, respectively. Relatively few splicing factors have been shown to be specific to the minor spliceosome. We found that the maize (Zea mays) RNA binding motif protein 48 (RBM48) is a U12 splicing factor that functions to promote cell differentiation and repress cell proliferation. RBM48 is coselected with the U12 splicing factor, zinc finger CCCH-type, RNA binding motif, and Ser/Arg rich 2/Rough endosperm 3 (RGH3). Protein-protein interactions between RBM48, RGH3, and U2 Auxiliary Factor (U2AF) subunits suggest major and minor spliceosome factors required for intron recognition form complexes with RBM48. Human RBM48 interacts with armadillo repeat containing 7 (ARMC7). Maize RBM48 and ARMC7 have a conserved protein-protein interaction. These data predict that RBM48 is likely to function in U12 splicing throughout eukaryotes and that U12 splicing promotes endosperm cell differentiation in maize.

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

Factor V Leiden (F5L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/- ). F8 deficiency enhanced the survival of F5L/LTfpi+/- mice, demonstrating that F5L/LTfpi+/- lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+Tfpi+/- females were crossed to generate 6,729 progeny, with 98 F5L/LTfpi+/- offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (MF5L1-16)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/- ) suppressed F5L/LTfpi+/- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/LTfpi+/- lethality (P = 1.7 × 10-6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.

The effects of turmeric (curcumin) on tumor suppressor protein (p53) and estrogen receptor (ERα) in breast cancer cells.

Curcumin (CUR) is a compound that has antibacterial, antiviral, anti-inflammatory, and anticancer properties. In this study, we have analyzed the effects of CUR on the expression of ERα and p53 in the presence of hormones and anti-hormones in breast cancer cells. Cells were cultured in a medium containing charcoal-stripped fetal bovine serum to deplete any endogenous steroids and treated with CUR at varying concentrations or in combination with hormones and anti-hormones. Protein analysis revealed a relative decrease in the levels of p53 and ERα upon treatment with 5-60 µM CUR. In cell proliferation studies, CUR alone caused a 10-fold decrease compared with the treatment with estrogen, which suggests its antiproliferative effects. Delineating the role of CUR in the regulation of p53, ERα, and their mechanisms of action may be important in understanding the influence of CUR on tumor suppressors and hormone receptors in breast cancer.

The regulation of tumor suppressor protein, p53, and estrogen receptor (ERα) by resveratrol in breast cancer cells.

Resveratrol (RES) is a natural antioxidant found abundantly in grapes, peanuts, and berries, and is known to possess anti-tumorigenic properties. However, there is a noticeable lack of studies on the mechanistic effects of Resveratrol on tumor suppressors. Previous studies from our laboratory have shown the tumor suppressor protein p53 and estrogen receptor-alpha (ERα) to be possible molecular targets for RES. In this study, the anti-estrogenic effects of RES were analyzed on the expression of ERα and p53. The breast cancer cells grown in stripped serum were treated with 60 µM RES, as the optimum concentration based on data obtained from a concentration study using 1-100 µM RES. Our studies indicate that RES caused a decrease in the levels of protein expression of p53 and ERα as compared to the control. Increasing concentrations of RES caused a four-fold decrease in cell number in comparison to estradiol. RES, in conjunction with ICI 182,780 (ICI), caused a down-regulation of both p53 and ERα as compared to the control. These observed effects on cell proliferation and regulation of both p53 and ERα by RES may lead to further understanding of the relationship between tumor suppressor proteins and steroid receptors in breast cancer cells.

Differential pre-mRNA Splicing Alters the Transcript Diversity of Helitrons Between the Maize Inbred Lines.

The propensity to capture and mobilize gene fragments by the highly abundant Helitron family of transposable elements likely impacts the evolution of genes in Zea mays. These elements provide a substrate for natural selection by giving birth to chimeric transcripts by intertwining exons of disparate genes. They also capture flanking exons by read-through transcription. Here, we describe the expression of selected Helitrons in different maize inbred lines. We recently reported that these Helitrons produce multiple isoforms of transcripts in inbred B73 via alternative splicing. Despite sharing high degrees of sequence similarity, the splicing profile of Helitrons differed among various maize inbred lines. The comparison of Helitron sequences identified unique polymorphisms in inbred B73, which potentially give rise to the alternatively spliced sites utilized by transcript isoforms. Some alterations in splicing, however, do not have obvious explanations. These observations not only add another level to the creation of transcript diversity by Helitrons among inbred lines but also provide novel insights into the cis-acting elements governing splice-site selection during pre-mRNA processing.

Effects of estrogen metabolite 2-methoxyestradiol on tumor suppressor protein p53 and proliferation of breast cancer cells.

An endogenous 17ß-estradiol (E(2)) metabolite, 2-methoxyestradiol (2-ME(2)), has been reported to exhibit estrogen receptor (ER)-independent anti-angiogenic and anti-tumor effects. Several mechanisms have been proposed for 2-ME(2) actions, but there is a lack of evidence for a common pathway for all of the cell-types sensitive to this metabolite. We have examined potential alterations in p53 in response to 2-ME(2), E(2) and the microtubule disruptor taxol in T47D breast cancer cells. Cells were cultured for six days in medium depleted of endogenous steroids or effectors. Semi-confluent cells were treated with 2-ME(2) (1 nM - 10 µM), 10 nM E(2) and/or 1 µM taxol and subjected to SDS-PAGE and Western blot analysis, quantitative analysis, or laser-scanning confocal microscopy. Western blot analysis revealed a concentration-dependent biphasic trend in p53 levels. Addition of 10 nM - 1 µM 2-ME(2) induced significant up-regulation in p53, and this response gradually diminished to levels comparable to the control upon treatment with higher concentrations (2.5 - 10 µM). The observed upregulation of p53 induced by 2-ME(2) is inhibited by concurrent treatment with 1 µM taxol. Cell quantitation revealed a significant decrease (50 - 90%) in cell number upon treatment with 1 - 10 µM 2-ME(2) with minimal effect at lower concentrations. No additional effect on cell proliferation was observed when taxol was combined with 10 nM or 1 µM 2-ME(2). In a concentration dependent manner, treatment with 2-ME(2) for 24 h differentially influenced cellular localization of p53. These results may aid in further understanding the relationship between steroid receptors, tumor suppressor proteins, and effects of hormone metabolites on breast cancer cells.